Antitumor immune response changes drastically during the progression of cancers. Established cancers often escape from the host immune system, although specific immune surveillance operates in the ...early stages of tumorigenesis in murine models. CD4+CD25+ regulatory T cells (TR) play a central role in self-tolerance and suppress effective antitumor immune responses. The aim of this study was to investigate the clinical significance and roles of TR in the progression and multistep carcinogenesis of pancreatic ductal adenocarcinoma.
We raised anti-FOXP3 antibodies and used them in immunohistochemical studies of the prevalence of FOXP3+CD4+CD25+ TR in the CD4+ T cells, which infiltrated in tissue and draining lymph nodes of 198 pancreatic ductal adenocarcinomas, their premalignant lesions (84 lesions of pancreatic intraepithelial neoplasias and 51 intraductal papillary-mucinous neoplasms), and 15 nonneoplastic pancreatic lesions.
The prevalence of TR was significantly increased in the ductal adenocarcinomas compared with that in the stroma of nonneoplastic inflammation (P<0.0001). The increased prevalence of T(R) was significantly correlated with certain clinicopathologic factors. A better prognosis was observed in patients with a low prevalence of T(R), and this was independent of other survival factors (P<0.0001). Infiltration of intraepithelial CD8+TIA-1+ cytotoxic T cells in pancreatic ducts was marked in low-grade premalignant lesions but diminished during the progression of both pancreatic intraepithelial neoplasias and intraductal papillary-mucinous neoplasms. Conversely, the prevalence of TR increased significantly during the progression of premalignant lesions.
T(R) play a role in controlling the immune response against pancreatic ductal carcinoma from the premalignant stage to established cancer. In pancreatic ductal carcinoma, a high prevalence of TR seems to be a marker of poor prognosis.
Cell‐cell adhesion determines the polarity of cells and participates in the maintenance of the cell societies called tissues. Cell‐cell adhesiveness is generally reduced in human cancers. Reduced ...intercellular adhesiveness allows cancer cells to disobey the social order, resulting in destruction of histological structure, which is the morphological hallmark of malignant tumors. Reduced intercellular adhesiveness is also indispensable for cancer invasion and metastasis. A tumor‐suppressor gene product, E‐cadherin, and its undercoat proteins, catenins, which connect cadherins to actin filaments, are located at lateral borders, concentrating on adherens junctions, of epithelial cells and establish firm cell‐cell adhesion. The E‐cadherin cell adhesion system in cancer cells is inactivated by various mechanisms that reflect the morphological and biological characteristics of the tumor. Silencing of the E‐cadherin gene by DNA hypermethylation around the promoter region occurs frequently, even in precancerous conditions. In diffuse infiltrating cancers, mutations are found in the genes for E‐cadherin and α‐and β‐catenins. At the invading front of cancers, the E‐cadherin cell adhesion system is inactivated by tyrosine phosphorylation of β‐catenin; an oncogene product, c‐erb B‐2 protein, is found to associate directly with β‐catenin. The E‐cadherin cell adhesion system cross‐talks with the Wingless/Wnt signaling pathway through β‐catenin, and expression of genes, which participate in cancer morphogenesis, may be regulated in conjunction with the Wingless/Wnt signaling pathway. Dysadherin, a newly identified cancer‐associated cell membrane glycoprotein, down‐regulates E‐cadherin and promotes cancer metastasis. In conclusion, inactivation of the E‐cadherin cell adhesion system by both genetic and epigenetic mechanisms plays a significant role during multistage human carcinogenesis.
It has long been known that cell-cell adhesiveness is generally reduced in human cancers. Tumor cells are dissociated throughout the entire tumor masses of diffuse-type cancers, whereas those of ...solid tumors with high metastatic potentials are often focally dissociated or dedifferentiated at the invading fronts. Thus, both irreversible and reversible mechanisms for inactivating the cell adhesion system appear to exist. This paper focuses on the cadherin system, which mediates Ca
2+-dependent homophilic cell-cell adhesion. The E (epithelial)-cadherin-mediated cell adhesion system in cancer cells is inactivated by multiple mechanisms corresponding to the pathological features described above. Mutations have been found in the genes for E-cadherin and its undercoat proteins, α- and β-catenins, which connect cadherins to actin filaments and establish firm cell-cell adhesion. Transcriptional inactivation of E-cadherin expression was shown to occur frequently in tumor progression. E-cadherin expression in human cancer cells is regulated by CpG methylation around the promoter region. The cadherin system interacts directly with products of oncogenes, eg, c-
erbB-2 protein and the epidermal growth factor receptor, and of the tumor suppressor gene, adenomatous polyposis coli (APC) protein, through β-catenin, which may be important in signal transduction pathways contributing to the determination of the biological properties of human cancers. In conclusion, inactivation of the E-cadherin system by multiple mechanisms, including both genetic and epigenetic events, plays a significant role in multistage carcinogenesis.
Background & Aims: Biliary tract cancer (BTC) is a highly malignant tumor, and identification of effective therapeutic targets to improve prognosis is urgently required. Oncogenic activation of ...survival genes is important for cancer cells to overcome oxidative stresses induced by their microenvironments that include chronic inflammation or exposure to anticancer drugs. We attempted to examine whether deregulation of Nrf2, a master transcriptional factor of various cytoprotective genes against oxidative stress, plays a role in the carcinogenesis of BTC. Methods: We screened genetic alteration of Keap1, a negative regulator of Nrf2, in BTC including tumors originated from gallbladder and extra- and intrahepatic bile ducts. Functional analysis of cancer-related mutant Keap1 in Nrf2 repression and the association between Nrf2 activation and resistance to 5-fluorouracil (5-FU) were investigated. Results: Recurrent (in 1/11 cell lines and 6/53 primary tumors) Keap1 gene alterations were observed in BTC and were especially frequent (4/13, 30.7%) in gallbladder cancer (GBC). These alterations led to a considerable loss of Nrf2 repression activity, caused constitutive activation of Nrf2, and promoted cell proliferation. Down-regulation of Nrf2 activity by either Keap1 complementation or Nrf2 short interference RNA increased sensitivity to 5-FU in Keap1-altered BTC cells. Conclusions: Keap1 mutation occurs frequently in GBC. Aberrant Nrf2 activation provoked by Keap1 alteration is one of the molecular mechanisms for chemotherapeutic resistance in GBC and will be a novel therapeutic target as an enhancer of sensitivity to 5-FU-based regimens.
Proteome data combined with histopathological information provides important, novel clues for understanding cancer biology and reveals candidates for tumor markers and therapeutic targets. We have ...established an application of a highly sensitive fluorescent dye (CyDye DIGE Fluor saturation dye), developed for two-dimensional difference gel electrophoresis (2D-DIGE), to the labeling of proteins extracted from laser microdissected tissues. The use of the dye dramatically decreases the protein amount and, in turn, the number of cells required for 2D-DIGE; the cells obtained from a 1 mm2 area of an 8-12 microm thick tissue section generate up to 5,000 protein spots in a large-format 2D gel. This protocol allows the execution of large-scale proteomics in a more efficient, accurate and reproducible way. The protocol can be used to examine a single sample in 5 d or to examine hundreds of samples in large-scale proteomics.
Wnt signaling inside the nucleus Shitashige, Miki; Hirohashi, Setsuo; Yamada, Tesshi
Cancer science,
April 2008, Volume:
99, Issue:
4
Journal Article
Peer reviewed
Open access
Accumulation of the β‐catenin protein and transactivation of a certain set of T‐cell factor (TCF)‐4 target genes by accumulated β‐catenin have been considered crucial in colorectal carcinogenesis. In ...the present review, we summarize nuclear proteins that interact with, and regulate, the β‐catenin and TCF and lymphoid enhancer factor (LEF) transcriptional complexes. Our recent series of proteomic studies has also revealed that various classes of nuclear proteins participate in the β‐catenin–TCF‐4 complex and modulate its transcriptional activity. Furthermore, the protein composition of the TCF‐4‐containing nuclear complex is not fixed, but is regulated dynamically by endogenous programs associated with intestinal epithelial cell differentiation and exogenous stimuli. Restoration of the loss‐of‐function mutation of the adenomatous polyposis coli (APC) gene in colorectal cancer cells does not seem to be a realistic approach with currently available medical technologies, and only signaling molecules downstream of the APC gene product can be considered as targets of pharmacological intervention. Nuclear proteins associated with the β‐catenin–TCF‐4 complex may include feasible targets for molecular therapy against colorectal cancer. Recently, an inhibitor of the interaction between CREB‐binding protein and β‐catenin was shown to efficiently shut down the transcriptional activity of TCF‐4 and induce apoptosis of colorectal cancer cells. We also summarize current strategies in the development of drugs against Wnt signaling. (Cancer Sci 2008; 99: 631–637)
The nuclear factor E2-related factor 2 (Nrf2) is a master transcriptional activator of genes encoding numerous cytoprotective enzymes that are induced in response to environmental and endogenously ...derived oxidative/electrophilic agents. Under normal, nonstressed circumstances, low cellular concentrations of Nrf2 are maintained by proteasomal degradation through a Keap1-Cul3-Roc1-dependent mechanism. A model for Nrf2 activation has been proposed in which two amino-terminal motifs, DLG and ETGE, promote efficient ubiquitination and rapid turnover; known as the two-site substrate recognition/hinge and latch model. Here, we show that in human cancer, somatic mutations occur in the coding region of NRF2, especially among patients with a history of smoking or suffering from squamous cell carcinoma; in the latter case, this leads to poor prognosis. These mutations specifically alter amino acids in the DLG or ETGE motifs, resulting in aberrant cellular accumulation of Nrf2. Mutant Nrf2 cells display constitutive induction of cytoprotective enzymes and drug efflux pumps, which are insensitive to Keap1-mediated regulation. Suppression of Nrf2 protein levels by siRNA knockdown sensitized cancer cells to oxidative stress and chemotherapeutic reagents. Our results strongly support the contention that constitutive Nrf2 activation affords cancer cells with undue protection from their inherently stressed microenvironment and anti-cancer treatments. Hence, inactivation of the Nrf2 pathway may represent a therapeutic strategy to reinforce current treatments for malignancy. Congruously, the present study also provides in vivo validation of the two-site substrate recognition model for Nrf2 activation by the Keap1-Cul3-based E3 ligase.
Oxidative and electrophilic stresses are sensed by Keap1, which activates Nrf2 to achieve cytoprotection by regulating the expression of drug-metabolizing and antioxidative stress enzymes/proteins. ...Because oxidative and electrophilic stresses cause many diseases, including cancer, we hypothesized that an abnormality in the Nrf2-Keap1 system may facilitate the growth of cancer cells. We sequenced the KEAP1 gene of 65 Japanese patients with lung cancer and identified five nonsynonymous somatic mutations at a frequency of 8%. We also identified two nonsynonymous somatic KEAP1 gene mutations and two lung cancer cell lines expressing KEAP1 at reduced levels. In lung cancer cells, low Keap1 activity (due to mutations or low-level expression) led to nuclear localization and constitutive activation of Nrf2. The latter resulted in constitutive expression of cytoprotective genes encoding multidrug resistance pumps, phase II detoxifying enzymes, and antioxidative stress enzymes/proteins. Up-regulation of these target genes in lung cancer cells led to cisplatin resistance. Nrf2 activation also stimulated growth of lung cancer-derived cell lines expressing KEAP1 at low levels and in mutant cell lines and in Keap1-null mouse embryonic fibroblasts under homeostatic conditions. Thus, inhibition of NRF2 may provide new therapeutic approaches in lung cancers with activation of Nrf2.
Tumor-infiltrating lymphocytes represent the host immune response to cancer. CD4+CD25+FOXP3+ regulatory T cells (Tregs) suppress the immune reaction. The aim of the present study was to investigate ...the clinicopathologic significance and roles of Tregs and CD8+ T cells during hepatocarcinogenesis.
We examined the infiltration of FOXP3+ Tregs and CD8+ T cells in the tumor stroma and nontumorous liver parenchyma using 323 hepatic nodules including precursor lesions, early hepatocellular carcinoma (HCC), and advanced HCC, along with 39 intrahepatic cholangiocarcinomas and 59 metastatic liver adenocarcinomas. We did immunohistochemical comparative studies.
The prevalence of Tregs was significantly higher in HCC than in the nontumorous liver (P<0.001). The patient group with a high prevalence of Tregs infiltrating HCC showed a significantly lower survival rate (P=0.007). Multivariate analysis revealed that the prevalence of Tregs infiltrating HCC was an independent prognostic factor. The prevalence of Tregs increased in a stepwise manner (P<0.001) and that of CD8+ T cells decreased during the progression of hepatocarcinogenesis (P<0.001). Regardless of the presence of hepatitis virus infection or histopathologic evidence of hepatitis, the prevalence of Tregs was significantly increased in nontumorous liver bearing primary hepatic tumors.
Tregs play a role in controlling the immune response to HCC during the progression of hepatocarcinogenesis. It has been suggested that primary hepatic cancers develop in liver that is immunosuppressed by a marked infiltration of Tregs. A high prevalence of Tregs infiltrating HCC is thought to be an unfavorable prognostic indicator.
In multicellular organisms, adaptive responses to oxidative stress are regulated by NF-E2-related factor 2 (NRF2), a master transcription factor of antioxidant genes and phase II detoxifying enzymes. ...Aberrant activation of NRF2 by either loss-of-function mutations in the Keap1 gene or gain-of-function mutations in the Nrf2 gene occurs in a wide range of human cancers, but details of the biological consequences of NRF2 activation in the cancer cells remain unclear. Here, we report that mutant NRF2 induces epithelial cell proliferation, anchorage-independent growth, and tumorigenicity and metastasis in vivo. Genome-wide gene expression profiling revealed that mutant NRF2 affects diverse molecular pathways including the mammalian target of rapamycin (mTOR) pathway. Mutant NRF2 upregulates RagD, a small G-protein activator of the mTOR pathway, which was also overexpressed in primary lung cancer. Consistently, Nrf2-mutated lung cancer cells were sensitive to mTOR pathway inhibitors (rapamycin and NVP-BEZ235) in both in vitro and an in vivo xenograft model. The gene expression signature associated with mutant NRF2 was a marker of poor prognosis in patients with carcinoma of the head and neck region and lung. These results show that oncogenic Nrf2 mutation induces dependence on the mTOR pathway during carcinogenesis. Our findings offer a rationale to target NRF2 as an anticancer strategy, and they suggest NRF2 activation as a novel biomarker for personalized molecular therapies or prognostic assessment.
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