Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide with high prevalence and lethality. However, the underlying mechanism for HCC has not been entirely elucidated. Recent studies have highlighted the roles of long non-coding RNAs (lncRNAs) in carcinogenesis, and it is suggested that they might play critical roles in HCC progression. Here, we will briefly introduce the biology of lncRNAs, emphasizing the mechanisms and emerging roles of HCC-related lncRNAs. To date, HCC-related lncRNAs are demonstrated to influence the life cycle of genes by various means including epigenetic silencing, splicing regulation, lncRNA–miRNA interaction, lncRNA–protein interaction and genetic variation. Moreover, they can participate in diverse biological processes involved in HCC progression through impacts upon cell proliferation, apoptosis, invasion and metastasis and angiogenesis. Since lncRNA can present in body fluid and have good specificity and accessibility, some HCC-related lncRNAs are suggested to be useful as novel potential biomarkers for HCC diagnosis, prognosis and prediction of response to therapy. Those HCC-related lncRNAs may provide potential novel therapeutic targets for HCC and other diseases.
Emerging evidence indicates that cancer is primarily a metabolic disease involving disturbances in energy production through respiration and fermentation. The genomic instability observed in tumor cells and all other recognized hallmarks of cancer are considered downstream epiphenomena of the initial disturbance of cellular energy metabolism. The disturbances in tumor cell energy metabolism can be linked to abnormalities in the structure and function of the mitochondria. When viewed as a mitochondrial metabolic disease, the evolutionary theory of Lamarck can better explain cancer progression than can the evolutionary theory of Darwin. Cancer growth and progression can be managed following a whole body transition from fermentable metabolites, primarily glucose and glutamine, to respiratory metabolites, primarily ketone bodies. As each individual is a unique metabolic entity, personalization of metabolic therapy as a broad-based cancer treatment strategy will require fine-tuning to match the therapy to an individual’s unique physiology.
Proteolytic maturation of various precursor proteins by the proprotein convertase Furin is now considered as a crucial step in tumor progression and metastasis. Here, we report the repression of the malignant and metastatic potential of carcinoma cells by the prodomain region of Furin (ppFurin), a naturally occurring inhibitor of this convertase. Overexpression of ppFurin in carcinoma cells in a stable manner significantly reduced their convertase activity and ability to mediate processing of the Furin cancer-related substrates platelet-derived growth factor (PDGF)-A and insulin-like growth factor-I receptor precursors. Unprocessed platelet-derived growth factor-A produced by ppFurin expressing cells failed to induce the activation of Akt in the platelet-derived growth factor receptor-expressing cells NIH BALB/c-3T3 and treatment of ppFurin expressing cells with insulin-like growth factor-I failed to induce Akt phosphorylation, compared with controls. The malignant potential of ppFurin expressing cells was significantly reduced as revealed by the loss of anchorage-independent growth and survival that associated their increased chemosensitivity. In vivo, comparative studies revealed that expression of ppFurin in the carcinoma cells MDA-MB-231 and CT-26 cells inhibited tumor growth when subcutaneously inoculated in nude mice. The use of an experimental liver colorectal metastasis model revealed the reduced ability of metastatic carcinoma CT-26 cells to colonize the liver in response to intrasplenic/portal inoculation. Further analyses revealed reduced Furin activity in tumors derived from intrasplenic inoculated mice with ppFurin expressing CT-26 cells. This finding highlights the role of Furin in the malignant and metastatic potential of tumor cells and suggests the possible consideration of using its naturally occurring inhibitor ppFurin in anticancer therapy.
Twist2 is a highly conserved basic helix-loop-helix transcription factor that plays a critical role in embryogenesis. Recent evidence has revealed that aberrant Twist2 expression contributes to tumor progression; however, the role of Twist2 in human hepatocellular carcinoma (HCC) and its underlying mechanisms remain undefined. In this report, we demonstrate that Twist2 is overexpressed in human HCC tumors. We show that ectopic expression of Twist2 induces epithelial–mesenchymal transition phenotypes, augments cell migration and invasion and colony-forming abilities in human HCC cells in vitro, and promotes tumor growth in vivo. Moreover, we found a higher percentage of CD24+ liver cancer stem-like cells in Twist2-transduced HCC cells. Twist2-expressing cells exhibited an increased expression of stem cell markers Bmi-1, Sox2, CD24 and Nanog and an increased capacity for self-renewal. Knockdown of CD24 in HepG2/Twist2 cells decreased the levels of Sox2, pSTAT3 and Nanog, and reversed the cancer stem-like cell phenotypes induced by ectopic expression of Twist2. Furthermore, Twist2 regulated the CD24 expression by directly binding to the E-box region in CD24 promoter. Therefore, our data demonstrated that Twist2 augments liver cancer stem-like cell self-renewal in a CD24-dependent manner. Twist2–CD24–STAT3–Nanog pathway may play a critical role in regulating liver cancer stem-like cell self-renewal. The identification of the Twist2-CD24 signaling pathway provides a potential therapeutic approach to target cancer stem cells in HCCs.
Epithelial ovarian cancer presents mostly with serous, endometrioid or mucinous histology but is treated as a single disease. The development of histotype-specific therapy has been challenging because of the relative lack of studies attributing disrupted pathways to a distinct histotype differentiation. mTOR activation is frequently associated with poor prognosis in serous ovarian cancer, which is the most common and most deadly histotype. However, the mechanisms dysregulating mTOR in the pathogenesis of ovarian cancer are unknown. We detected copy number loss and correlated lower expression levels of LKB1, TSC1, TSC2 and PTEN tumor suppressor genes for upstream regulators of mTOR activity in up to 80% in primary ovarian serous tumor databases, with LKB1 allelic loss-predominant. Reduced LKB1 protein was usually associated with increased mTOR activity in both serous ovarian cancer cell lines and primary tumors. Conditional deletion of Lkb1 in murine ovarian surface epithelial (OSE) cells caused papillary hyperplasia and shedding but not tumors. Simultaneous deletion of Lkb1 and Pten, however, led to development of high-grade ovarian serous histotype tumors with 100% penetrance that expressed WT1, ERα, PAX8, TP53 and cytokeratin 8, typical markers used in the differential diagnosis of serous ovarian cancer. Neither hysterectomy nor salpingectomy interfered with progression of ovarian tumorigenesis, suggesting that neither uterine nor Fallopian tube epithelial cells were contributing to tumorigenesis. These results implicate LKB1 loss in the OSE in the pathogenesis of serous ovarian cancer and provide a compelling rationale for investigating the therapeutic potential of targeting LKB1 signaling in patients with this deadly disease.
MicroRNA deregulation and pathway alterations have been implicated in nasopharyngeal carcinoma (NPC), a highly invasive and metastatic cancer widely prevalent in Southern China. In this study, we report that miR-9 is commonly downregulated in NPC specimens and NPC cell lines with important functional consequences. The reduced expression of miR-9 was inversely correlated with clinical stages and marked the progression from locoregional to metastatic tumors. The CpG island hypermethylation contributed to miR-9 silencing in NPC cell lines and tissues. Ectopic expression of miR-9 dramatically inhibited the proliferative, migratory and invasive capacities of NPC cells in vitro and in vivo. We found that miR-9 strongly reduced the expression of CXCR4 in NPC cells. Luciferase assay demonstrated that miR-9 could directly bind to the 3' untranslated region of CXCR4. Similar to the restoring miR-9 expression, CXCR4 downregulation inhibited cell growth, migration and invasion, whereas CXCR4 overexpression rescued the suppressive effect of miR-9. Mechanistic investigations revealed that CXCR4 functionally mediated the SDF-1-stimulated activation of p38 mitogen-activated protein kinase pathway in NPC cells with miR-9 downregulation or CXCR4 overexpression. In clinical specimens, CXCR4 and phospho-p38 were widely overexpressed, and the levels increased with the progression from locoregional to metastatic tumors in NPC tissues. The levels of CXCR4 were inversely correlated with miR-9 or phospho-p38 expression. Taken together, our results indicate that miR-9 functions as a tumor-suppressive microRNA in NPC, and that its suppressive effects are mediated chiefly by repressing CXCR4 expression.
The O6-methylguanine-DNA methyltransferase gene (MGMT) encodes the direct reversal DNA repair protein that removes alkyl adducts from the O6 position of guanine. Several single-nucleotide polymorphisms (SNPs) exist in the MGMT promoter/enhancer (P/E) region. However, the haplotype structure encompassing these SNPs and their functional/biological significance are currently unknown. We hypothesized that MGMT P/E haplotypes, rather than individual SNPs, alter MGMT transcription and can thus alter human sensitivity to alkylating agents. To identify the haplotype structure encompassing the MGMT P/E region SNPs, we sequenced 104 DNA samples from healthy individuals and inferred the haplotypes using the data generated. We identified eight SNPs in this region, namely T7C (rs180989103), T135G (rs1711646), G290A (rs61859810), C485A (rs1625649), C575A (rs113813075), G666A (rs34180180), C777A (rs34138162) and C1099T (rs16906252). Phylogenetics and Sequence Evolution analysis predicted 21 potential haplotypes that encompass these SNPs ranging in frequencies from 0.000048 to 0.39. Of these, 10 were identified in our study population as 20 paired haplotype combinations. To determine the functional significance of these haplotypes, luciferase reporter constructs representing these haplotypes were transfected into glioblastoma cells and their effect on MGMT promoter activity was determined. Compared with the most common (reference) haplotype 1, seven haplotypes significantly upregulated MGMT promoter activity (18–119% increase; P < 0.05), six significantly downregulated MGMT promoter activity (29–97% decrease; P < 0.05) and one haplotype had no effect. Mechanistic studies conducted support the conclusion that MGMT P/E haplotypes, rather than individual SNPs, differentially regulate MGMT transcription and could thus play a significant role in human sensitivity to environmental and therapeutic alkylating agents.
Lung cancer is the leading cause of cancer-related deaths worldwide. By now, genome-wide association studies (GWAS) have identified numerous loci associated with the risk of developing lung cancer. However, these loci account for only a small fraction of the familial lung cancer risk. We hypothesized that epistasis may contribute to the missing heritability. To test this hypothesis, we systematically evaluated the association of epistasis of genetic variants with risk of lung cancer in Han Chinese cohorts. We conducted a pairwise genetic interaction analysis of 591370 variants, using BOolean Operation-based Screening and Testing (BOOST), in an ongoing GWAS of lung cancer that includes 2331 cases and 3077 controls. Pairs of epistatic loci with PBOOST ≤ 1.00x10–6 were further evaluated by a logistic regression model (LRM) with covariate adjustment. Four promising epistatic pairs identified at the screening stage (PLRM ≤ 2.86x10–13) were validated in two replication cohorts: the first from Beijing (1534 cases and 1489 controls) and the second from Shenyang and Guangzhou (2512 cases and 2449 controls). Using this combined analysis, we identified an interaction between rs2562796 and rs16832404 at 2p32.2 that was significantly associated with the risk of developing lung cancer (PLRM = 1.03x10–13 in total 13 392 subjects). This study is the first investigation of epistasis for lung cancer on a genome-wide scale in Han Chinese. It addresses part of the missing heritability in lung cancer risk and provides novel insight into the multifactorial etiology of lung cancer.
Dozens of common genetic variants associated with cancer risk have been identified through genome-wide association studies (GWASs). However, these variants only explain a modest fraction of the heritability of disease. The missing heritability has been attributed to several factors, among them the existence of genetic interactions (G x G). Systematic screens for G x G in model organisms have revealed their fundamental influence in complex phenotypes. In this scenario, G x G overlap significantly with other types of gene and/or protein relationships. Here, by integrating predicted G x G from GWAS data and complex- and context-defined gene coexpression profiles, we provide evidence for G x G associated with cancer risk. G x G predicted from a breast cancer GWAS dataset identified significant overlaps [relative enrichments (REs) of 8–36%, empirical P values < 0.05 to 10–4] with complex (non-linear) gene coexpression in breast tumors. The use of gene or protein data not specific for breast cancer did not reveal overlaps. According to the predicted G x G, experimental assays demonstrated functional interplay between lipoma-preferred partner and transforming growth factor-β signaling in the MCF10A non-tumorigenic mammary epithelial cell model. Next, integration of pancreatic tumor gene expression profiles with pancreatic cancer G x G predicted from a GWAS corroborated the observations made for breast cancer risk (REs of 25–59%). The method presented here can potentially support the identification of genetic interactions associated with cancer risk, providing novel mechanistic hypotheses for carcinogenesis.
Mutation of p53 is a common feature of cancer. Breast cancer is the most common malignancy that develops in women; however, somatic mutation of p53 is rare, suggesting that p53 becomes inactivated by other mechanisms. p53 is expressed as smaller isoforms, some of which inhibit wild-type p53. There are no studies that have examined the relative expression of all isoforms in this disease. We have analysed the relative messenger RNA expression of the p53 isoforms, 40, 133, β and in a panel of 6 breast cancer cell lines, 148 breast cancers specimens and 31 matched normal adjacent tissues by semi-quantitative real-time reverse transcription–PCR and analysed their relationship to clinical features and outcome. We have identified several important clinical associations, particularly with 40p53, which was expressed at levels that were ~50-fold higher than the least expressed isoform p53. 40p53 was significantly upregulated in tumour tissue when compared with the normal breast and was significantly associated with an aggressive breast cancer subtype—triple negative. Additionally, p53β expression was significantly negatively associated with tumour size and positively associated with disease-free survival, where high levels of p53β were protective, particularly in patients with a mutation in p53, suggesting p53β may counteract the damage inflicted by mutant p53. In conclusion, the relative expression of p53 isoforms is related to clinical features of breast cancer and outcome. These results have implications for the stratification of breast cancer based on p53 function and may provide an alternate explanation for deregulated p53 signalling in breast cancer.
CXCL5 is a member of the CXC-type chemokine family that may play a role in carcinogenesis and cancer progression. This study investigates the biological function and clinical significance of CXCL5 in intrahepatic cholangiocarcinoma (ICC). We demonstrated that CXCL5 was overexpressed in ICC cell lines and tumor samples compared with paired normal tissues. CXCL5 had a direct chemoattractant effect on neutrophils in vitro through PI3K-Akt and extracellular signal-regulated kinase 1/2 signaling pathways. In animal studies, CXCL5 promoted tumor growth and metastasis without altering in vitro proliferative and invasive ability of ICC cells, and this effect was mediated by the recruitment of intratumoral infiltrative neutrophils by tumor-derived CXCL5. Immunohistochemical analysis of ICC samples showed that overexpression of CXCL5 correlated strongly with intratumoral neutrophil infiltration, shorter overall survival and high tumor recurrence. Multivariate analysis revealed that CXCL5 overexpression alone, or combined with the presence of intratumoral neutrophils, was an independent prognostic indicator for ICC. In conclusion, our data showed that CXCL5 promotes ICC growth and metastasis by recruiting intratumoral neutrophils. CXCL5 alone or combined with intratumoral neutrophils is a novel prognostic predictor for ICC patients and a potential therapeutic target.
Arachidonic acid (20:45,8,11,14, AA)-derived prostaglandin E2 (PGE2) promotes colon cancer development. In contrast, chemoprotective n-3 polyunsaturated fatty acids supplant AA, thereby decreasing PGE2 biosynthesis in colonocytes, with eicosapentaenoic acid (20:55,8,11,14,17, EPA) in particular being metabolized to a novel 3-series E-prostaglandin (PGE3), a putative anti-tumorigenic-cyclooxygenase metabolite. Because transformation of adult stem cells is an extremely important route toward initiating intestinal cancer, we utilized the leucine-rich-repeat-containing G-protein-coupled receptor 5 (Lgr5)-enhanced green fluorescent protein-internal ribosome entry site (IRES)-creERT2 knock-in mouse model to isolate and culture colonic organoids, in order to document ex vivo responses to exogenous PGE2 and PGE3. Colonic crypts were isolated from transgenic mice and cultured in a Matrigel-based three-dimensional platform. Organoids were treated with exogenous PGE2, PGE3 or dimethyl sulfoxide (vehicle control) for 5 days and the number of viable organoids was recorded daily. Subsequently, samples were processed for immunohistochemistry, flow cytometry and real-time PCR analyses. PGE2 promoted optimal organoid growth and induced significantly higher levels of cell proliferation (P < 0.05) compared with PGE3 and control. In contrast, the Lgr5-green fluorescent protein-positive stem cell number was uniquely elevated by >2-fold in PGE2-treated cultures compared with PGE3 and control. This coincided with the upregulation of stem-cell-related Sox9, Axin2 and Cd44 messenger RNAs. Our results demonstrate that relative to AA-derived PGE2, a known promoter of colon tumorigenesis, EPA-derived PGE3 has diminished ability to support colonic stem cell expansion in mouse colonic organoids.
Previous studies have implicated cancer stem cells in tumor recurrence and revealed that the stem cell gene SOX2 plays an important role in the tumor cell resistance to apoptosis. Nonetheless, the mechanism by which SOX2 regulates apoptosis signals remained undefined. Here, we demonstrated the surprising finding that silencing of the SOX2 gene effectively induces apoptosis via the activation of death receptor and mitochondrial signaling pathways in human non-small cell lung cancer cells. Unexpectedly, reverse transcription–PCR analysis suggested that downregulation of SOX2 leads to activation of MAP4K4, previously implicated in cell survival. Evaluation of the apoptotic pathways revealed an increased expression of key inducers of apoptosis, including tumor necrosis factor-α and p53, with concurrent attenuation of Survivin. Although p53 appeared dispensable for this pathway, the loss of Survivin in SOX2-deficient cells appeared critical for the observed MAP4K4 induced cell death. Rescue experiments revealed that SOX2-silencing-mediated killing was blocked by ectopic expression of Survivin, or by reduction of MAP4K4 expression. Clinically, expressions of Survivin and SOX2 were highly correlated with each other. The results reveal a key target of SOX2 expression and highlight the unexpected context-dependent role for MAP4K4, a pluripotent activator of several mitogen-activated protein kinase pathways, in regulating tumor cell survival.
SH3RF (SH3-domain-containing RING finger protein) family members, SH3RF1-3, are multidomain scaffold proteins involved in promoting cell survival and apoptosis. In this report, we show that SH3RF2 is an oncogene product that is overexpressed in human cancers and regulates p21-activated kinase 4 (PAK4) protein stability. Immunohistochemical analysis of 159 colon cancer tissues showed that SH3RF2 expression levels are frequently elevated in cancer tissues and significantly correlate with poor prognostic indicators, including increased invasion, early recurrence and poor survival rates. We also demonstrated that PAK4 protein is degraded by the ubiquitin–proteasome system and that SH3RF2 inhibits PAK4 ubiquitination via physical interaction-mediated steric hindrance, which results in the upregulation of PAK4 protein. Moreover, ablation of SH3RF2 expression attenuates TRADD (TNFR-associated death domain) recruitment to tumor necrosis factor-α (TNF-α) receptor 1 and hinders downstream signals, thereby inhibiting NF-B (nuclear factor-kappaB) activity and enhancing caspase-8 activity, in the context of TNF-α treatment. Notably, ectopic expression of SH3RF2 effectively prevents apoptosis in cancer cells and enhances cell migration, colony formation and tumor growth in vivo. Taken together, our results suggest that SH3RF2 is an oncogene that may be a definitive regulator of PAK4. Therefore, SH3RF2 may represent an effective therapeutic target for cancer treatment.
The multifunctional E4F1 protein is a cellular target of the E1A adenoviral oncoprotein. Interaction between E4F1 and the hepatitis B virus (HBV) protein HBx has been demonstrated in vitro. In this study, RNA interference has been used to downregulate E4F1 in the hepatocellular carcinoma (HCC) cell line HepG2 (HBV negative) and its derivative, HBV expressing HepG2/2.2.15. Reduction of E4F1 levels induced hepatocyte vacuolation (formation of large cytoplasmic vesicles), increased autophagy and caused mitochondrial defects and metabolism changes in HepG2/2.2.15, but not in HepG2. Moreover, downregulation of E4F1 reduced DNA synthesis with partial cell cycle arrest in G1 in both cell types and this effect was more marked in HepG2/2.2.15 than in HepG2. These effects were partially prevented by RNA interference directed to either HBx or to p53. Coprecipitation and western blot experiments detected complexes between E4F1 and HBx in several HCC cell lines. Although a review of mutation and gene expression public databases did not support that E4F1 is specifically altered in liver cancer, our results suggest that E4F1 may neutralize the capacity of HBx to activate a p53-dependent, metabolic and growth arrest phenotype in liver cells, thus possibly contributing to the viability and proliferation of HBV-infected cells.
Epidemiological studies showed that women have a lower bladder cancer (BCa) incidence, yet higher muscle-invasive rates than men, suggesting that estrogen and the estrogen receptors, estrogen receptor alpha (ERα) and estrogen receptor beta (ERβ), may play critical roles in BCa progression. Using in vitro cell lines and an in vivo carcinogen N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN)-induced mouse BCa model, we found that ERβ plays a positive role in promoting BCa progression. Knockdown of ERβ with ERβ-shRNA in ERβ-positive human BCa J82, 647v and T24 cell lines led to suppressed cell growth and invasion. Mice lacking ERβ have less cancer incidence with reduced expression of the proliferation marker Ki67 in BBN-induced BCa. Consistently, our results show that non-malignant urothelial cells with ERβ knockdown are more resistant to carcinogen-induced malignant transformation. Mechanism dissection found that targeting ERβ suppressed the expression of minichromosome maintenance complex component 5 (MCM5), a DNA replication licensing factor that is involved in tumor cell growth. Restoring MCM5 expression can partially reverse ERβ knockdown-mediated growth reduction. Supportively, treating cells with the ERβ-specific antagonist, 4-[2-Phenyl-5,7-bis(trifluoromethyl) pyrazolo[1,5-a]pyrimidin-3-yl]phenol (PHTPP), reduced BCa cell growth and invasion, as well as MCM5 expression. Furthermore, we provide the first evidence that BCa burden and mortality can be controlled by PHTPP treatment in the carcinogen-induced BCa model. Together, these results demonstrate that ERβ could play positive roles in promoting BCa progression via MCM5 regulation. Targeting ERβ through ERβ-shRNA, PHTPP or via downstream targets, such as MCM5, could serve as potential therapeutic approaches to battle BCa.
Oral squamous cell carcinoma (SCC) is among the most prevalent cancers in the world and is characterized by high morbidity and few therapeutic options. Like most cancers, oral SCC arises from a multistep process involving alterations of genes responsible for balancing proliferation and differentiation. Among these, Krppel-like factor 4 (Klf4) suppresses cell proliferation and promotes differentiation and thus helps to maintain epithelial homeostasis. However, the prevailing role of Klf4 in maintenance of normal homeostasis in oral epithelium has not been established in vivo. Here, we used an inducible oral-specific mice model to selectively ablate Klf4 in the oral cavity. We generated K14-CreERTam/Klf4f/f mice that survived to adulthood and did not present overt phenotype. However, histologically these mice showed dysplastic lesions, increased cell proliferation and abnormal differentiation in the tongue 4 months after induction, supporting a homeostatic role of Klf4 in the oral epithelia. Furthermore, by breeding these mutants with a transgenic line expressing at endogenous levels K-rasG12D, we assessed the role of disrupting differentiation gene programs to the carcinogenesis process. The K14-CreERTAM/K-rasG12D/Klf4–/– mice rapidly develop oral SCC in the tongue. Thus, our findings support the emerging notion that activation of differentiating gene programs may represent a barrier preventing carcinogenesis in epithelial cells harboring oncogenic mutations, and thus that molecules acting upstream and downstream of Klf4 may represent components of a novel tumor-suppressive pathway.
RNA interference has boosted the field of functional genomics, by making it possible to carry out ‘loss-of-function’ screens in cultured cells. Here, we performed a small interfering RNA screening, in three breast cancer cell lines, for 101 candidate driver genes overexpressed in amplified breast tumors and belonging to eight amplicons on chromosomes 8q and 17q, investigating their role in cell survival/proliferation. This screening identified eight driver genes that were amplified, overexpressed and critical for breast tumor cell proliferation or survival. They included the well-described oncogenic driver genes for the 17q12 amplicon, ERBB2 and GRB7. Four of six other candidate driver genes—RAD21 and EIF3H, both on chromosome 8q23, CHRAC1 on chromosome 8q24.3 and TANC2 on chromosome 17q23—were confirmed to be driver genes regulating the proliferation/survival of clonogenic breast cancer cells presenting an amplification of the corresponding region. Indeed, knockdown of the expression of these genes decreased cell viability, through both cell cycle arrest and apoptosis induction, and inhibited the formation of colonies in anchorage-independent conditions, in soft agar. Strategies for inhibiting the expression of these genes or the function of the proteins they encode are therefore of potential value for the treatment of breast cancers presenting amplifications of the corresponding genomic region.
Non-small cell lung cancer (NSCLC) is the leading cause of cancer mortality worldwide. Despite progress in developing chemotherapeutics for the treatment of NSCLC, primary and secondary resistance limits therapeutic success. NSCLC cells exhibit multiple mutations in the epidermal growth factor receptor (EGFR), which cause aberrant activation of diverse cell signaling pathways. Therefore, suppression of the inappropriate amplification of EGFR downstream signaling cascades is considered to be a rational therapeutic and preventive strategy for the management of NSCLC. Our initial molecular target–oriented virtual screening revealed that the ginger components, including -shogaol, -paradol and -gingerol, seem to be potential candidates for the prevention and treatment of NSCLC. Among the compounds, -shogaol showed the greatest inhibitory effects on the NSCLC cell proliferation and anchorage-independent growth. -Shogaol induced cell cycle arrest (G1 or G2/M) and apoptosis. Furthermore, -shogaol inhibited Akt kinase activity, a downstream mediator of EGFR signaling, by binding with an allosteric site of Akt. In NCI-H1650 lung cancer cells, -shogaol reduced the constitutive phosphorylation of signal transducer and activator of transcription-3 (STAT3) and decreased the expression of cyclin D1/3, which are target proteins in the Akt signaling pathway. The induction of apoptosis in NCI-H1650 cells by -shogaol corresponded with the cleavage of caspase-3 and caspase-7. Moreover, intraperitoneal administration of -shogaol inhibited the growth of NCI-H1650 cells as tumor xenografts in nude mice. -Shogaol suppressed the expression of Ki-67, cyclin D1 and phosphorylated Akt and STAT3 and increased terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling-positivity in xenograft tumors. The current study clearly indicates that -shogaol can be exploited for the prevention and/or treatment of NSCLC.
The alcohol aversion drug disulfiram (DSF) reacts and conjugates with the protein-bound nucleophilic cysteines and is known to elicit anticancer effects alone or improve the efficacy of many cancer drugs. We investigated the effects of DSF on human O6-methylguanine-DNA methyltransferase (MGMT), a DNA repair protein and chemotherapy target that removes the mutagenic O6-akyl groups from guanines, and thus confers resistance to alkylating agents in brain tumors. We used DSF, copper-chelated DSF or CuCl2–DSF combination and found that all treatments inhibited the MGMT activity in two brain tumor cell lines in a rapid and dose-dependent manner. The drug treatments resulted in the loss of MGMT protein from tumor cells through the ubiquitin-proteasome pathway. Evidence showed that Cys145, a reactive cysteine, critical for DNA repair was the sole site of DSF modification in the MGMT protein. DSF was a weaker inhibitor of MGMT, compared with the established O6-benzylguanine; nevertheless, the 24–36h suppression of MGMT activity in cell cultures vastly increased the alkylation-induced DNA interstrand cross-linking, G2/M cell cycle blockade, cytotoxicity and the levels of apoptotic markers. Normal mice treated with DSF showed significantly attenuated levels of MGMT activity and protein in the liver and brain tissues. In nude mice bearing T98 glioblastoma xenografts, there was a preferential inhibition of tumor MGMT. Our studies demonstrate a strong and direct inhibition of MGMT by DSF and support the repurposing of this brain penetrating drug for glioma therapy. The findings also imply an increased risk for alkylation damage in alcoholic patients taking DSF.
Beta-naphthoflavone (BNF, DB06732) is an agonist of aryl hydrocarbon receptor (AhR) and a putative chemotherapeutic agent that has antitumor activity against mammary carcinomas in vivo. However, the mechanism by which BNF exerts this antitumor effect remains unclear. Thus, we explored mechanisms of BNF’s antitumor effects in human breast cancer cells. This study showed that BNF suppressed cell proliferation and induced cell cycle arrest in the G0/G1 phase with downregulation of cyclin D1/D3 and CDK4 and upregulation of p21Cip1/Waf1, leading to a senescence-like phenotype in estrogen receptor (ER)-positive MCF-7 cells, but not in ER-negative MDA-MB-231 cells. In addition, BNF inhibited PI3K/AKT signaling, and the PI3K inhibitor, LY294,002, exhibited the same inhibitory effects on cyclinD1/D3, CDK4 and the cell cycle as BNF. Interestingly, BNF activated mitogen-activated protein kinase-extracellular signal-regulated kinase (MAPK-ERK) signaling, and more notably, MEK inhibitor PD98059 significantly blocked the BNF-induced cell cycle arrest and upregulation of p21Cip1/Waf1. Furthermore, specific ERα and AhR siRNA studies indicate that ERα is required in BNF-induced p21Cip1/Waf1 expression, and BNF-mediated cell cycle arrest and modulation of AKT and ERK signaling is AhR-dependent. Taken together, AhR-dependent inhibition of the PI3K/AKT pathway, activation of MAPK/ERK and modulation of ERα is a novel mechanism underlying BNF-mediated antitumor effects in breast cancer, which may represent a promising strategy to be exploited in future clinical trials.
Despite advances in detection and treatment for breast cancer (BC), recurrence and death rates remain unacceptably high. Therefore, more convenient diagnostic and prognostic methods still required to optimize treatments among the patients. Here, we report the clinical significance of the serum cathepsin E (CatE) activity as a novel prognostic marker for BC. Correlation analysis between the serum levels of CatE expression and clinicopathological parameters revealed that the activity levels, but not the protein levels, were negatively associated with the stages and progression of BC. Univariate and multivariate analyses demonstrated that the serum CatE activity was significantly correlated with favorable prognostic outcomes of the patients. The functional link of CatE expression to BC progression was further corroborated by in vivo and in vitro studies with mice exhibiting different levels of CatE expression. Multiparous CatE–/– mice spontaneously developed mammary tumors concomitant with morphological transformation and altered growth characteristics of the mammary glands. These alterations were associated in part with the induction of epithelial–mesenchymal transition and the activation of β-catenin-dependent pathway in mammary cells. Loss of CatE strongly induced the translocation and accumulation of Wnt5a in the nuclei, thereby leading to the aberrant trafficking, maturation and secretion of Wnt5a and the impaired signaling. The interaction of CatE and Wnt5a was verified by proximity ligation assay and by knockdown or restoration of CatE expression in the mammary cells. Consequently, our data demonstrate that CatE contributes to normal growth and development of mammary glands through proper trafficking and secretion of Wnt5a.
Inter-α-trypsin inhibitor heavy chain 5 (ITIH5) has been associated with tumour suppression in various cancers. However, its putative role in bladder cancer is completely unknown. Therefore, we initiated a study analysing ITIH5 expression as well as its prognostic and functional impact on human urothelial cancers (UCs). Expression analysis showed a clear down-regulation of ITIH5 mRNA in 61% (n = 45) of UCs, especially in muscle-invasive tumours (P < 0.001). ITIH5 loss in UCs was further evident on protein level (65.5%, n = 55) as detected by immunohistochemistry. DNA methylation analysis demonstrated tumour-specific ITIH5 promoter methylation in 50% of papillary none-invasive pTa (n = 30) and 68% of invasive (n = 28) UCs. Aberrant ITIH5 promoter methylation in bladder tumours was tightly linked (P < 0.001) with loss of ITIH5 mRNA expression, which was furthermore functionally confirmed by demethylation analysis in cell lines. Pyrosequencing analysis revealed that ITIH5 promoter hypermethylation was closely associated with progressive bladder cancers. Subsequently, a large cohort (n = 120) of clinically challenging pT1 high-grade UC was analysed for ITIH5 expression. Of clinical significance, we found an association between loss of ITIH5 expression and unfavourable prognosis of UC patients without distant metastasis at first diagnosis (recurrence-free survival; hazard ratio: 4.35, P = 0.048). Functionally, ITIH5 re-expression in human RT112 bladder cancer cells led to both suppression of cell migration and inhibition of colony spreading. Hence, we provide evidence that down-regulation of ITIH5 by aberrant DNA hypermethylation may provoke invasive phenotypes in human bladder cancer. Moreover, ITIH5 protein might become a prognostic biomarker for relapse risk stratification in high-grade UC patients.