Previous studies have shown that dormant licensed replication origins can be exploited to enhance recovery from replication stress. Since tumour cells express high levels of origin licensing proteins, we examined whether depletion of such factors might specifically sensitise tumour versus non-tumour cells. Consistent with previous findings, we observed that three tumour-derived cell lines overexpress ORC1, a licensing component, compared to four non-tumour cell lines and that a greater level of ORC1 was required to maintain viability in the tumour cells. We determined siRNA-mediated knockdown conditions for each line that maximally reduced ORC1 but did not impact upon viability, which we considered would optimally deplete dormant origins. ORC1 depletion hypersensitised the tumour-derived cells to hydroxyurea (HU) and H202 but did not affect the sensitivity of the non-tumour lines. Similar results were observed following depletion of ORC6 or CDC6. Further, co-depletion of p53 and ORC1 modestly impaired viability of 1BR3hTERT non-tumour fibroblasts and more dramatically caused hypersensitivity to HU. Finally, overexpression of the c-Myc oncogene combined with ORC1 depletion in non-tumour BJhTERT cells diminished viability. Collectively, these findings suggest that tumour cells may have a reliance on origin licensing capacity, suggesting that licensing factors could represent a target for drug-based cancer therapy.
Origin licensing; ORC1; replication stress; tumour cell lines; c-Myc overexpression
Cell- and receptor-specific regulation of cell migration by Gi/oα-proteins remains unknown in prostate cancer cells. In the present study, oxytocin (OXT) receptor (OXTR) was detected at the protein level in total cell lysates from C81 (an androgen-independent subline of LNCaP), DU145 and PC3 prostate cancer cells, but not in immortalized normal prostate luminal epithelial cells (RWPE1), and OXT induced migration of PC3 cells. This effect of OXT has been shown to be mediated by Gi/oα-dependent signaling. Accordingly, OXT inhibited forskolin-induced luciferase activity in PC3 cells that were transfected with a luciferase reporter for cAMP activity. Although mRNAs for all three Giα isoforms were present in PC3 cells, Giα2 was the most abundant isoform that was detected at the protein level. Pertussis toxin (PTx) inhibited the OXT-induced migration of PC3 cells. Ectopic expression of the PTx-resistant Giα2-C352G, but not wild type Giα2, abolished this effect of PTx on OXT-induced cell migration. The Giα2-targeting siRNA was shown to specifically reduce Giα2 mRNA and protein in prostate cancer cells. The Giα2-targeting siRNA eliminated OXT-induced migration of PC3 cells. These data suggest that Giα2 plays an important role in the effects of OXT on PC3 cell migration. The Giα2-targeting siRNA also inhibited EGF-induced migration of PC3 and DU145 cells. Expression of the siRNA-resistant Giα2, but not wild type Giα2, restored the effects of EGF in PC3 cells transfected with the Giα2-targeting siRNA. In conclusion, Giα2 plays an essential role in OXT and EGF signaling to induce prostate cancer cell migration.
Epidermal growth factor; Oxytocin receptor; G-protein; Prostate cancer; Cell migration
Almost half of the human genome and as much as 40% of the mouse genome is composed of repetitive DNA sequences. The majority of these repeats are retrotransposons of the SINE and LINE families, and such repeats are generally repressed by epigenetic mechanisms. It has been proposed that these elements can act as methylation centers from which DNA methylation spreads into gene promoters in cancer. Contradictory to a methylation center function, we have found that retrotransposons are enriched near promoter CpG islands that stay methylation-free in cancer. Clearly, it is important to determine which influence, if any, these repetitive elements have on nearby gene promoters. Using an in vitro system, we confirm here that SINE B1 elements can influence the activity of downstream gene promoters, with acquisition of DNA methylation and loss of activating histone marks, thus resulting in a repressed state. SINE sequences themselves did not immediately acquire DNA methylation, but were marked by H3K9me2 and H3K27me3. Moreover, our bisulfite sequencing data did not support that gain of DNA methylation in gene promoters occurred by methylation spreading from SINE B1 repeats. Genome-wide analysis of SINE repeats distribution showed that their enrichment is directly correlated with the presence of USF1, USF2 and CTCF binding, proteins with insulator function. In summary, our work supports the concept that SINE repeats interfere negatively with gene expression and that their presence near gene promoters is counter-selected, except when the promoter is protected by an insulator element.
DNA methylation; gene silencing; retrotransposons
TGF-β plays a dual role in epithelial carcinogenesis with the potential to either suppress or promote tumor progression. We found that levels of Smad3 mRNA, a critical mediator of TGF-β signaling, are reduced by ~60% in human breast cancer. We therefore used conditionally immortalized mammary epithelial cells (IMECs) of differing Smad3 genotypes to quantitatively address the Smad3 requirement for different biological responses to TGF-β. We found that a two-fold reduction in Smad3 gene dosage led to complex effects on TGF-β responses; the growth inhibitory response was retained, the pro-apoptotic response was lost, the migratory response was reduced and the invasion response was enhanced. Loss of the pro-apoptotic response in the Smad3+/- IMECs correlated with loss of Smad3 binding to the Bcl-2 locus, while retention of the growth inhibitory response in Smad3+/- IMECs correlated with retention of Smad3 binding to the c-Myc locus. Addressing the integrated outcome of these changes in vivo, we showed that reduced Smad3 levels enhanced metastasis in two independent models of metastatic breast cancer. Our results suggest that different biological responses to TGF-β in the mammary epithelium are differentially affected by Smad3 dosage, and that a mere two-fold reduction in Smad3 is sufficient to promote metastasis.
The epidermal growth factor receptor (EGFR) contributes to tumor radioresistance, in part, through interactions with the catalytic subunit of DNA-dependent Protein Kinase (DNA-PKcs), a key enzyme in the non homologous end joining DNA repair pathway. We previously demonstrated that EGFR-DNA-PKcs interactions are significantly compromised in the context of activating mutations in EGFR in non small cell lung carcinoma (NSCLC) and human bronchial epithelial cells. Here, we investigate the reciprocal relationship between phosphorylation status of DNA-PKcs and EGFR-mediated radiation response. The data reveal that both the kinase activity of DNA-PKcs and radiation-induced phosphorylation of DNA-PKcs by the Ataxia Telangiectasia Mutated (ATM) kinase are critical prerequisites for EGFR-mediated radioresponse. Alanine substitutions at 7 key serine/threonine residues in DNA-PKcs or inhibition of DNA-PKcs by NU7441 completely abrogated EGFR-mediated radioresponse and blocked EGFR binding. ATM-deficiency or ATM inhibition with KU55933 produced a similar effect. Importantly, alanine substitution at an ATM-dependent DNA-PKcs phosphorylation site, T2609, was sufficient to block binding or radioresponse of EGFR. However, mutation of a DNA-PKcs auto-phosphorylation site, S2056 had no such effect indicating that DNA-PKcs auto-phosphorylation is not necessary for EGFR-mediated radioresponse. Our data reveal that in both NSCLCs and HBECs, activating mutations in EGFR specifically abolished the DNA-PKcs phosphorylation at T2609, but not S2056. Our study underscores the critical importance of a reciprocal relationship between DNA-PKcs phosphorylation and EGFR mediated radiation response and elucidates mechanisms underlying mutant EGFR associated radiosensitivity in NSCLCs.
The aryl hydrocarbon receptor (AHR) has been shown to play a role in an increasing number of cellular processes. Recent reports have linked the AHR to cell proliferation, cytoskeletal arrangement, and tumor invasiveness in various tumor cell types. The AHR plays a role in the de-repression of the IL6 promoter in certain tumor cell lines, allowing for increased transcriptional activation by cytokines. Here, we show that there is a significant level of constitutive activation of the AHR in cells isolated from head and neck squamous cell carcinoma (HNSCC) patients. Constitutive activation of the AHR in HNSCC cells was blocked by antagonist treatment, leading to a reduction in IL6 expression. Additionally, the AHR exhibits a high level of expression in HNSCC cells compared to normal keratinocytes. These findings led to the hypothesis that the basal AHR activity in HNSCC cells plays a role in the aggressive phenotype of these tumors, and that antagonist treatment could mitigate this phenotype. This study provides evidence that antagonism of the AHR in HNSCC tumor cells, in the absence of exogenous receptor ligands, has a significant effect on tumor cell phenotype. Treatment of these cell lines with the AHR antagonists 6, 2′, 4′-trimethoxyflavone, or the more potent GNF351, decreased migration, and invasion of HNSCC cells and prevented benzo[a]pyrene-mediated induction of the chemotherapy efflux protein ABCG2. Thus, an AHR antagonist treatment has been shown to have therapeutic potential in HNSCC through a reduction in aggressive cell phenotype.
Ah receptor; dioxin; antagonist; head and neck; HNSCC; cancer
Tumor proteases and inhibitors have been associated with paradoxical effects on tumor progression in preclinical and clinical settings. We previously reported that urokinase (uPA) overexpression delays tumor progression in mammary cancer. This study aimed to determine the role of plasminogen activator inhibitor-1 (PAI-1) on uPA’s paradoxical in vivo effects. Using syngeneic murine models, we found that stable uPA overexpression promoted in vivo growth of colon tumors (MC-38) naturally expressing high PAI-1, while growth inhibition was observed in renal tumors (RENCA) expressing lower PAI-1 levels. In murine mammary carcinoma (4T1), uPA overexpression shifted the uPA/PAI-1 balance in favor of the protease, resulting in significantly reduced tumor growth and metastases in vivo. Conversely, increased tumor progression was observed in stable PAI-1 overexpressing 4T1 tumors, compared to uPA overexpressing and control tumors. These effects were associated with down regulation of metastases promoting genes in uPA overexpressing tumors, such as metalloproteinases, CXCL-1, c-Fos, integrin α-5, VEGF-A, PDGF-α and IL-1β. In PAI-1 overexpressing tumors, many of the above genes were upregulated. PAI-1 overexpressing tumors had increased total and new tumor microvessels, and increased tumor cell proliferation, while the opposite effects were found in uPA overexpressing tumors. Finally, PAI-1 down-regulation led to significant inhibition of 4T1 tumor growth and metastases in vivo. In conclusion, uPA’s dual effects on tumor progression occur in the context of its interactions with endogenous PAI-1 expression. Our studies uncover novel mechanisms of in vivo tumor control by modulation of the balance between tumor proteases and inhibitors, which may be exploited therapeutically.
Taxol (paclitaxel) is a potent anticancer drug that has been found to be effective against several tumor types, including cervical cancer. However, the exact mechanism underlying the antitumor effects of paclitaxel is poorly understood. Here, paclitaxel induced the apoptosis of cervical cancer HeLa cells and correlated with the enhanced activation of caspase-3 and TAp73, which was strongly inhibited by TAp73β small interfering RNA (siRNA). In wild-type activating transcription factor 3 (ATF3)–overexpressed cells, paclitaxel enhanced apoptosis through increased α and β isoform expression of TAp73; however, these events were attenuated in cells containing inactive COOH-terminal–deleted ATF3 [ATF3(ΔC)] or ATF3 siRNA. In contrast, paclitaxel-induced ATF3 expression did not change in TAp73β -overexpressed or TAp73β siRNA–cotransfected cells. Furthermore, paclitaxel-induced ATF3 translocated into the nucleus where TAp73β is expressed, but not in ATF3 (ΔC) or TAp73β siRNA–transfected cells. As confirmed by the GST pull-down assay, ATF3 bound to the DNA-binding domain of p73, resulting in the activation of p21 or Bax transcription, a downstream target of p73. Overexpression of ATF3 prolonged the half-life of TAp73β by inhibiting its ubiquitination and thereby enhancing its transactivation and proapoptotic activities. Additionally, ATF3 induced by paclitaxel potentiated the stability of TAp73β, not its transcriptional level. Chromatin immunoprecipitation analyses show that TAp73β and ATF3 are recruited directly to the p21 and Bax promoter. Collectively, these results reveal that overexpression of ATF3 potentiates paclitaxel-induced apoptosis of HeLa cells, at least in part, by enhancing TAp73β's stability and its transcriptional activity. The investigation shows that ATF3 may function as a tumor-inhibiting factor through direct regulatory effects on TAp73β, suggesting a functional link between ATF3 and TAp73β.
MicroRNAs are small noncoding RNAs that have critical roles in regulating a number of cellular functions through transcriptional silencing. They have been implicated as oncogenes and tumor suppressor genes (oncomirs) in several human neoplasms. We used an integrated genomics and functional screening strategy to identify potential oncomirs in the pediatric neoplasm neuroblastoma. We first identified microRNAs that map within chromosomal regions that we and others have defined as frequently deleted (1p36, 3p22, and 11q23-24) or gained (17q23) in high-risk neuroblastoma. We then transiently transfected microRNA precursor mimics or inhibitors into a panel of six neuroblastoma cell lines that we characterized for these genomic aberrations. The majority of transfections showed no phenotypic effect, but the miR-34a (1p36) and miR-34c (11q23) mimics showed dramatic growth inhibition in cell lines with 1p36 hemizygous deletion. In contrast, there was no growth inhibition by these mimics in cell lines without 1p36 deletions. Quantitative reverse transcription-PCR showed a perfect correlation of absent miR-34a expression in cell lines with a 1p36 aberration and phenotypic effect after mimetic add-back. Expression of miR-34a was also decreased in primary tumors (n = 54) with 1p36 deletion (P = 0.009), but no mutations were discovered in resequencing of the miR-34a locus in 30 neuroblastoma cell lines. Flow cytometric time series analyses showed that the likely mechanism of miR-34a growth inhibition is through cell cycle arrest followed by apoptosis. BCL2 and MYCN were identified as miR-34a targets and likely mediators of the tumor suppressor phenotypic effect. These data support miR-34a as a tumor suppressor gene in human neuroblastoma.
Group I p21-activated kinases (PAKs) are important effectors of the small GTPases Rac and Cdc42, which regulate cell motility/migration, survival, proliferation and gene transcription. Hyperactivation of these kinases have been reported in many tumor types, making PAKs attractive targets for therapeutic intervention. PAKs are activated by growth factor-mediated signaling and are negatively regulated by the tumor suppressor NF2/Merlin. Thus, tumors characterized by NF2 inactivation would be expected to show hyperactivated PAK signaling. Based on this rationale, we evaluated the status of PAK signaling in malignant mesothelioma (MM), an aggressive neoplasm that is resistant to current therapies and shows frequent inactivation of NF2. We demonstrate that group I PAKs are activated in most MMs and MM cell lines and that genetic or pharmacological inhibition of PAKs is sufficient to inhibit MM cell proliferation and survival. We also identify downstream effectors and signaling pathways that may contribute mechanistically to PAK-related tumorigenesis. Specifically, we show that inhibition of PAK results in attenuation of AKT and Raf-MAPK signaling and decreased tumor cell viability. Collectively, these data suggest that pharmacological inhibition of group I PAKs may have therapeutic efficacy in tumors characterized by PAK activation.
p21-activated kinase (PAK); malignant mesothelioma; small molecule inhibitor; targeted therapy; NF2/Merlin; AKT; mitogen-activated protein kinases (MAPK); Raf-1
Increased inducible nitric oxide synthase (NOS2) expression in breast tumors is associated with decreased survival of estrogen receptor-negative (ER−) breast cancer patients. We recently communicated the preliminary observation that nitric oxide (NO) signaling results in EGFR tyrosine phosphorylation. To further define the role of NO in the pathogenesis of ER− breast cancer, we examined the mechanism of NO-induced EGFR activation in human ER− breast cancer. NO was found to activate EGFR and Src by a mechanism that includes S-nitrosylation. NO, at physiologically relevant concentrations, induced an EGFR/Src-mediated activation of oncogenic signal transduction pathways (including c-Myc, Akt and β-catenin) and the loss of PP2A tumor suppressor activity. Additionally, NO signaling increased cellular EMT, expression and activity of COX-2 and chemoresistance to adriamycin and paclitaxel. When connected into a network, these concerted events link NO to the development of a stem cell-like phenotype, resulting in the upregulation of CD44 and STAT3 phosphorylation. Our observations are also consistent with the finding that NOS2 is associated with a basal-like transcription pattern in human breast tumors. These results indicate that the inhibition of NOS2 activity or NO signaling networks may have beneficial effects in treating basal-like breast cancer patients.
S-Nitrosylation; nitric oxide; inducible nitric oxide synthase; basal-like; breast cancer
The Hedgehog (Hh) pathway has emerged as an important pathway in multiple tumor types and is thought to be dependent on a paracrine signaling mechanism. The purpose of this study was to determine the role of pancreatic cancer-associated fibroblasts (human pancreatic stellate cells, HPSCs) in Hh signaling. In addition, we evaluated the efficacy of a novel Hh antagonist, AZD8542, on tumor progression with an emphasis on the role of the stroma compartment.
Expression of Hh pathway members and activation of the Hh pathway were analyzed in both HPSCs and pancreatic cancer cells. We tested the effects of SMO inhibition with AZD8542 on tumor growth in vivo using an orthotopic model of pancreatic cancer containing varying amounts of stroma.
HPSCs expressed high levels of SMO receptor and low levels of Hh ligands, whereas cancer cells showed the converse expression pattern. HPSC proliferation was stimulated by sonic Hedgehog with upregulation of downstream GLI1 mRNA. These effects were abrogated by AZD8542 treatment. In an orthotopic model of pancreatic cancer, AZD8542 inhibited tumor growth only when HPSCs were present, implicating a paracrine signaling mechanism dependent on stroma. Further evidence of paracrine signaling of the Hh pathway in prostate and colon cancer models is provided, demonstrating the broader applicability of our findings.
Based on the use of our novel human derived pancreatic cancer stellate cells, our results suggest that Hh-targeted therapies primarily affect the tumor-associated stroma, rather than the epithelial compartment.
Pancreatic cancer; Tumor-stromal cell interactions; pancreatic stellate cells; Hedgehog
Glycogen Synthase Kinase-3 (GSK-3) is a serine/threonine kinase involved in a diverse range of cellular processes. GSK-3 exists in two isoforms, GSK-3α and GSK-3β, which possess some functional redundancy but also play distinct roles depending on developmental and cellular context. In this report we found that GSK-3 actively promoted cell growth and survival in melanoma cells, and blocking this activity with small molecule inhibitor SB216763 or gene-specific siRNA decreased proliferation, increased apoptosis and altered cellular morphology. These alterations coincided with loss of PAX3, a transcription factor implicated in proliferation, survival and migration of developing melanoblasts. We further found that PAX3 directly interacted with and was phosphorylated in vitro on a number of residues by GSK-3β. In melanoma cells, direct inhibition of PAX3 lead to cellular changes that paralleled the response to GSK-3 inhibition. Maintenance of PAX3 expression protected melanoma cells from the anti-tumor effects of SB216763. These data support a model wherein GSK-3 regulates proliferation and morphology of melanoma through phosphorylation and increased levels of PAX3.
Glycogen; Synthase; Kinase-3/melanoma/PAX3
Ewing's sarcoma is an aggressive pediatric cancer of the bone and soft tissue, in which patients whose tumors have a poor histological response to initial chemotherapy have a poor overall prognosis. Therefore, it is important to identify molecules involved in resistance to chemotherapy. Herein, we demonstrate that the DNA-repair protein and transcriptional cofactor, EYA3, is highly expressed in Ewing's sarcoma tumor samples and cell lines compared with mesenchymal stem cells, the presumed cell of origin of Ewing's sarcoma, and that it is regulated by the EWS/FLI1 fusion protein transcription factor. We further demonstrate that EWS/FLI1 mediates upregulation of EYA3 via repression of miR-708, a microRNA that targets the EYA3 3′UTR, rather than by binding the EYA3 promoter directly. Importantly, we demonstrate that high levels of EYA3 significantly correlate with low levels of miR-708 in Ewing's sarcoma samples, suggesting that this miR-mediated mechanism of EYA3 regulation holds true in human cancers. Because EYA proteins are important for cell survival during development, we examine, and demonstrate, that loss of EYA3 decreases survival of Ewing's sarcoma cells. Most importantly, knockdown of EYA3 in Ewing's sarcoma cells leads to sensitization to DNA-damaging chemotherapeutics used in the treatment of Ewing's sarcoma, and as expected, after chemotherapeutic treatment, EYA3 knockdown cells repair DNA damage less effectively than their control counterparts. These studies identify EYA3 as a novel mediator of chemoresistance in Ewing's sarcoma and define the molecular mechanisms of both EYA3 overexpression and of EYA3-mediated chemoresistance.
Ewing's sarcoma; EYA; microRNA; miR-708; chemoresistance
Pancreatic cancer significantly affects the quality of life due to the severe abdominal pain. However, the underlying mechanism is not clear. This study aimed to determine the relationship between substance P (SP) and pancreatic cancer perineural invasion (PNI) as well as mechanism of SP mediating pancreatic cancer PNI which cause pain in patients with pancreatic cancer. Human pancreatic cancer cells MIA PaCa-2, BxPC-3 and newborn dorsal root ganglions (DRGs) were used to determine the expression of SP or NK-1R in pancreatic cancer cells and DRGs cells by QT-PCR and Western blotting. The effects of SP on pancreatic cancer cell proliferation and invasion were analyzed using MTT assay and Transwell matrigel invasion assay, respectively. Alterations in the neurotropism of pancreatic cancer cells were assessed by co-culture system which mimics the interaction of tumor/neuron in vivo. SP is not only widely distributed in the neurite outgrowth from newborn DRGs but also expressed in MIA PaCa-2 and BxPC-3 cells. NK-1R is found to be overexpressed in the pancreatic cancer cell lines MIA PaCa-2 and BxPC-3. SP induces cancer cell proliferation and invasion and the expression of MMP-2 in pancreatic cancer cells; and NK-1R antagonists inhibit these effects. Furthermore, SP is also able to promote neurite outgrowth and the migration of pancreatic cancer cell cluster to the DRGs, which is blocked by NK-1R antagonists in the co-culture model. Our results suggest that SP plays an important role in the development of pancreatic cancer metastasis and PNI, and blocking the SP/NK-1R signaling system is a novel strategy for the treatment of pancreatic cancer.
Substance P (SP); NK-1 receptor (NK-1R); pancreatic cancer; perineural invasion (PNI)
MYC-induced DNA damage is exacerbated in WRN deficient cells, leading to replication stress and accelerated cellular senescence. To determine if WRN deficiency impairs MYC driven tumor development, we utilized both xenograft and autochthonous tumor models. Conditional silencing of WRN expression in c-MYC overexpressing non-small cell lung cancer xenografts impaired both tumor establishment and tumor growth. This inhibitory effect of WRN knock-down was accompanied by increased DNA damage, decreased proliferation, and tumor necrosis. In the Eμ-Myc mouse model of B-cell lymphoma, a germline mutation in the helicase domain of Wrn (WrnΔhel/Δhel) resulted in a significant delay in emergence of lethal lymphomas, extending tumor free survival by >30%. Analysis of pre-neoplastic B cells from Eμ-Myc Wrn mutant mice revealed increased DNA damage, elevation of senescence markers, and decreased proliferation in comparison with cells from age-matched Eμ-Myc mice. Immunohistochemical and global gene expression analysis of overt Eμ-Myc WrnΔhel/Δhel lymphomas demonstrated a marked increase in expression of the CDK inhibitor, p16Ink4a, as well as elevation of TAp63, a known mediator of senescence. Collectively, these studies demonstrate that in the context of Myc-associated tumorigenesis, loss of Wrn amplifies the DNA damage response, both in pre-neoplastic and neoplastic tissue, engaging activation of tumor suppressor pathways. This leads to inhibition of tumor growth and prolonged tumor free survival. Targeting WRN or its enzymatic function could prove to be an effective strategy in the treatment of MYC-associated cancers.
Werner helicase; therapeutic target; Myc-driven cancer; senescence; tumor suppressors
Squamous cell cancers comprise the most common types of human epithelial cancers. One subtype, head and neck squamous cell carcinoma (HNSCC), is a particularly aggressive cancer with poor prognosis due to late diagnosis and lymph node metastasis. Of all the processes involved in carcinogenesis, local invasion and distant metastasis are clinically the most relevant, but are the least well understood on a molecular level. Here we find that in vivo, the α-catenin homologue – α-catulin, a protein originally reported to interact with Lbc Rho guanine nucleotide exchange factor, is highly expressed at the tumor invasion front and in the metastatic streams of cells in both malignant human HNSCCs and a mouse model of oral SCC. Knockdown of α-catulin in hHNSCC cell lines dramatically decrease the migratory and invasive potential of those cells in vitro and metastatic potential in xenotransplants in vivo. Analysis of tumors deficient in α-catulin showed that the tumor cells are unable to invade the surrounding stroma. Accordingly, transcriptional profiling of those tumors revealed that α-catulin ablation is accompanied by changes in genes involved in cell migration and invasion. Interestingly enough, in vitro experiments show that an upregulation of α-catulin expression correlates with the transition of tumor cells from an epithelial to a mesenchymal morphology, as well as an upregulation of EMT markers vimentin and snail. Overall, these results strongly indicate that α-catulin contributes to the invasive behavior of metastatic cells, and may be used as a prognostic marker and future therapeutic target for cancer patients.
α-catulin; metastasis; invasion; EMT; squamous cell carcinoma
Studies suggest that Hsf4 expression correlates with its role in cell growth and differentiation. However, the role of Hsf4 in tumorigenesis in vivo remains unexplored. In this article, we provide evidence that absence of the Hsf4 gene suppresses evolution of spontaneous tumors arising in p53- or Arf-deficient mice. Furthermore, deletion of hsf4 alters the tumor spectrum by significantly inhibiting development of lymphomas that are normally observed in the majority of mice lacking p53 or Arf tumor suppressor genes. Using mouse embryo fibroblasts deficient in the hsf4 gene, we have found that these cells exhibit reduced proliferation that is associated with induction of senescence and senescence-associated β-galactosidase (SA-β-gal). Cellular senescence in hsf4-deficient cells is associated with the increased expression of the cyclin-dependent kinase inhibitors, p21 and p27 proteins. Consistent with the cellular senescence observed in vitro, specific normal tissues of hsf4−/− mice and tumors that arose in mice deficient in both hsf4 and p53 genes exhibit increased SA-β-gal activity and elevated levels of p27 compared with wild-type mice. These results suggest that hsf4 deletion-induced senescence is also present in vivo. Our results therefore indicate that Hsf4 is involved in modulation of cellular senescence, which can be exploited during cancer therapy.
The Eph receptor tyrosine kinases and ephrin ligands have been studied extensively for their roles in developmental processes. In recent years, Eph receptors and ephrins have been found to be integral players in cancer formation and progression. Among these are EphA2 and ephrinA1, which are involved in the development and maintenance of many different types of solid tumors. The function of EphA2 and ephrinA1 in tumorigenesis and tumor progression is complex and seems to be dependent on cell type and microenvironment. These variables affect the expression of the EphA2 and ephrinA1 proteins, the pathways through which they induce signaling, and the functional consequences of that signaling on the behavior of tumor cells and tumor-associated cells. This review will specifically focus on the roles that EphA2 and ephrinA1 play in the different cell types that contribute to the malignancy of solid tumors, with emphasis on the opportunities for therapeutic targeting.
Alterations in the microenvironment collaborate with cell autonomous mutations during the transformation process. Indeed, cancer- associated fibroblasts (CAF) and senescent fibroblasts stimulate tumorigenesis in xenograft models. Because senescent fibroblasts accumulate with age, these findings suggest that they contribute to age-related increases in tumorigenesis. Previously we demonstrated that senescent-associated stromal derived osteopontin (OPN) contributes to preneoplastic cell growth in vitro and in xenografts, suggesting that it impacts neoplastic progression. Analysis of fibroblasts within premalignant and malignant skin lesions ranging from solar/actinic keratosis (AK) to squamous cell carcinoma (SCC) revealed they express OPN. Given the stromal expression of OPN, we investigated how OPN impacts preneoplastic cell growth. We demonstrate that OPN promotes preneoplastic keratinocyte cellular proliferation and cell survival through the CD44 cell receptor and activation of the MAPK pathway. These data suggest that stromal-derived OPN impacts tumorigenesis by stimulating preneoplastic cell proliferation thus allowing expansion of initiated cells in early lesions.
stroma; microenvironment; osteopontin; senescence; preneoplastic
Metastasis is the final stage of tumor progression and is thought to be responsible for up to 90% of deaths associated with solid tumors. Caveolin-1 (CAV1) regulates multiple cancer-associated processes related to malignant tumor progression. In the present study we tested the hypothesis that CAV1 modulates the metastatic ability of ESFT cells. First, we analyzed the expression of CAV1 by immunostaining a tissue microarray containing 43 paraffin-embedded ESFT tumors with known EWS translocations. Even though no evidence was found for a significant association between CAV1 expression and stage, size or tumor site, all metastatic samples (10/10) had significantly high CAV1 expression, suggesting that high CAV1 content could positively contribute to enhance ESFT metastasis. To determine the effect of CAV1 on the migratory and invasive capabilities of ESFT cells, we knocked down CAV1 expression in TC252 and A673 cells by stably transfecting a previously validated shRNA construct. In vitro, migration and invasion assays showed that, for both cell lines CAV1 knocked-down cells migrated and invaded significantly less (p≤0.01) than control cells. Moreover, control A673 cells introduced into Balb/c nude mice by tail vein injection strongly colonized the lungs. In contrast, animals injected with CAV1 knocked-down cells showed either no incidence of metastasis or developed lung metastases after a significant delay (P<0.0001). Finally, we show that the molecular mechanisms by which CAV1 carries out its key role in regulating ESFT metastasis involve MMP production and activation as well as the control of the expression of SPARC, a known determinant of lung colonization.
The p38 MAPK pathway regulates multiple physiological and pathological processes, including cancer development. PRAK, a p38 substrate protein kinase, has previously been implicated in the suppression of skin carcinogenesis. In the current study, we demonstrate that PRAK deletion accelerates hematopoietic cancer development in a mouse model harboring an oncogenic ras allele, Eμ-N-RasG12D, specifically expressed in hematopoietic cells. Further investigation reveals that enhanced hematopoietic tumorigenesis by PRAK deficiency is associated with hyper-activation of the JNK pathway both in vivo and in primary hematopoietic cells isolated from spleens. In primary splenocytes, PRAK deficiency further enhanced oncogenic ras-induced cell proliferation, and promoted ras-mediated colony formation on semi-solid medium in a JNK-dependent manner. In addition, deletion of PRAK leads to abrogation of ras-induced accumulation of senescence markers. These findings indicate that PRAK suppresses hematopoietic cancer formation in this mouse model by antagonizing oncogenic ras-induced activation of the JNK pathway. Our results suggest that PRAK may function as a tumor suppressor in multiple types of cancers.
PRAK; ras; JNK; lymphomagenesis; oncogene-induced senescence; mouse cancer model
The Aurora-A kinase gene is frequently amplified and/or over-expressed in a variety of human cancers, leading to major efforts to develop therapeutic agents targeting this pathway. Here we demonstrate that Aurora-A is targeted for ubiquitination and subsequent degradation by the F-box protein FBXW7 in a process that is regulated by GSK3β. Using a series of truncated Aurora-A proteins and site directed mutagenesis, we identified distinct FBXW7 and GSK3β binding sites in Aurora-A. Mutation of critical residues in either site substantially disrupts degradation of Aurora-A. Furthermore, we show that loss of Pten results in the stabilization of Aurora-A by attenuating FBXW7-dependent degradation of Aurora-A through the AKT/GSK3β pathway. Moreover, radiation-induced tumor latency is significantly shortened in Fbxw7+/− Pten+/− mice as compared to either Fbxw7+/− or Pten+/− mice, indicating that Fbxw7 and Pten appear to cooperate in suppressing tumorigenesis. Our results establish a novel posttranslational regulatory network in which the Pten and Fbxw7 pathways appear to converge on the regulation of Aurora-A level.
Aurora-A; Fbxw7; Pten; tumorigenesis; Ubiquitination
The receptor tyrosine kinase Axl is over-expressed in a variety of cancers and is known to play a role in proliferation and invasion. Previous data from our lab indicates that Axl and its ligand GAS6 may play a role in establishing metastatic dormancy in the bone marrow microenvironment. In the current study, we found that Axl is highly expressed in metastatic prostate cancer (PCa) cell lines PC3 and DU145 and has negligible levels of expression in a non-metastatic cancer cell line LNCaP. Knockdown of Axl in PC3 and DU145 cells resulted in decreased expression of several mesenchymal markers including Snail, Slug, and N-cadherin, and enhanced expression of the epithelial marker E-cadherin, suggesting that Axl is involved in the epithelial to mesenchymal transition in PCa cells. The Axl-knockdown PC3 and DU145 cells also displayed decreased in vitro migration and invasion. Interestingly, when PC3 and DU145 cells were treated with GAS6, Axl protein levels were down-regulated. Moreover, CoCl2, a hypoxia mimicking agent, prevented GAS6 mediated down-regulation of Axl in these cell lines. Immunochemical staining of human PCa tissue microarrays demonstrated that Axl, GAS6 and Hif1-α (indicator of hypoxia) were all co-expressed in PCa and in bone metastases, compared to normal tissues. Together, our studies indicate that Axl plays a crucial role in PCa metastasis, and that GAS6 regulates the expression of Axl. Importantly, in a hypoxic tumor microenvironment Axl expression maintained leading to enhanced signaling.
Bone Metastasis; prostate cancer; EMT; Hypoxia; tumor microenvironment; GAS6; Axl
The immediate early response gene X-1 (IEX-1) is involved in regulation of various cellular processes including proliferation, apoptosis in part by controlling homeostasis of reactive oxygen species (ROS) at mitochondria. The present study demonstrates reduced inflammatory responses and colorectal cancer in IEX-1 knockout (KO) mice treated with azoxymethane (AOM)/dextran sulfate sodium (DSS). However, DSS induced worse colitis in RAG−/−IEX-1−/− double KO mice than in RAG and IEX-1 single KO mice, underscoring an importance of T cells in IEX-1 deficiency-induced protection against colon inflammation. Lack of IEX-1 promoted the differentiation of IL-17-producing T cells, concomitant with up regulation of Gαi2 expression, a gene that is well-documented for its role in the control of inflammation in the colon. In accordance with this, Th17 cell differentiation was compromised in the absence of Gαi2, and deletion of Gαi2 in T cells alone aggravated colon inflammation and colorectal cancer development after AOM/DSS treatment. Null mutation of IEX-1 also enhanced both proliferation and apoptosis of intestinal epithelial cells (IECs) after injury. A potential impact of this altered IEC turnover on colon inflammation and cancer development is discussed. These observations provide a linkage of IEX-1 and Gαi2 expression in the regulation of Th17 differentiation and suggest a previously unappreciated role for IEX-1 in the control of colon epithelial homeostasis.