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Tumor necrosis factor-α (TNF-α) is involved in epithelial-mesenchymal transition (EMT) and expression of CD44, a cancer stem cell marker, in several cancers. This study was performed to clarify the significance of TNF-α and CD44 in clear cell renal cell carcinomas (ccRCCs). Expression of TNF-α and CD44 was examined by immunohistochemistry in 120 ccRCCs. Involvement of TNF-α in EMT and induction of CD44 was analyzed by monitoring expression of EMT-related genes and CD44, and invasion in cultured ccRCC cell lines. TNF-α and CD44 were immunolocalized mainly to carcinoma cells of high-grade ccRCCs with positive correlations with primary tumor stage. A positive correlation was also obtained between TNF-α and CD44 expression, and co-upregulation of TNF-α and CD44 was associated with primary tumor stage, distant metastasis, and poor prognosis. TNF-α enhanced migration and invasion of ccRCC cells together with down-regulation of E-cadherin expression and up-regulation of matrix metalloproteinase 9 and CD44 expression. TNF-α also up-regulated the expression of TNF-α itself in ccRCC cells. Among the 25 ccRCC patients treated with sunitinib for metastatic disease, high CD44 expression was associated with poor treatment outcome. Importantly, residual carcinoma cells in the sunitinib-treated metastatic ccRCCs were strongly positive for CD44, and the CD44 expression was significantly higher in the tumors from the sunitinib-treated patients than in those from untreated ones. Our data show that TNF-α plays an important role in progression of ccRCCs by inducing EMT and CD44 expression, and suggest that CD44 induced by TNF-α may be involved in the resistance to the sunitinib treatment., (© 2014 UICC.)

Heterogeneity of tumor tissue has been accounted for in recent years by a hierarchy-based model in which cancer stem cells (CSCs) have the ability both to self-renew and to give rise to differentiated tumor cells and are responsible for the overall organization of a tumor. Research into CSCs has progressed rapidly and concomitantly with recent advances in the biology of normal tissue stem cells, resulting in the identification of CSCs in a wide range of human tumors. Studies of mouse models of human cancer have provided further insight into the characteristics of CSCs as well as a basis for the development of novel therapies targeted to these cells. However, recent studies have revealed complexities, such as plasticity of stem cell properties and clonal diversity of CSCs, in certain tumor types that have led to revision of the original CSC model. In this review, we summarize the history of the discovery and characterization of CSCs, as well as address recent advances that have revealed the complexity of these cells and their therapeutic implications., (Copyright © 2012 UICC.)

Triple-negative breast cancer [TNBC, which is negative for the estrogen receptor (ER), progesterone receptor, and human epidermal growth factor receptor 2] is a high-risk form of the disease without a specific therapy. DNA microarray and immunohistochemical analyses have shown that most TNBCs fall within the basal-like histological subset of breast cancers, which frequently exhibit inactivation of the retinoblastoma tumor suppressor (Rb) and upregulation of the cyclin-dependent kinase inhibitor p16(INK4a) (p16). However, downregulation of p16 expression has been observed in some basal-like breast cancer cell lines, suggesting that such cells can be divided into two groups according to Rb and p16 status. We now show that cells that are CD44(+) and CD24(-) , a phenotype associated with stem-like breast cancer cells, are more abundant in ER(-) /p16(-) breast cancer cell lines than in ER(-) /p16(+) lines. It was also found that p16 expression was downregulated in mammospheres from an ER-negative breast cancer cell line. Depletion of p16 by RNA interference in ER-negative breast cancer cells increased the percentage of CD44(+) /CD24(-) cells and increased the expression of mRNA of the ES-like genes Nanog, Oct4, and Sox2 through an Rb-independent pathway. Furthermore, such depletion of p16 reduced chemosensitivity. The loss of p16 expression may thus reduce the response of ER-negative breast cancer cells to chemotherapy by conferring cancer stem cell-like properties. Consistent with this conclusion, immunohistochemical analysis of the clinical samples suggests that low p16 expression in TNBC is associated with resistance to preoperative chemotherapy., (Copyright © 2011 UICC.)

The protein kinase A regulatory subunit 1 alpha (PRKAR1A/PKAI) pathway is overexpressed in varieties of tumors and cancer cell lines including cholangiocarcinoma (CCA), although its role in CCA growth modulation is unclear. In our study, we evaluated the effect of PRKAR1A/PKAI targeting on CCA cell proliferation. Real-time PCR demonstrated an increased mRNA expression of PRKAR1A/PKAI, whereas protein kinase A regulatory subunit 2 beta (PRKAR2B/PKAII) was downregulated in human CCA tissues and CCA cell lines. Immunohistochemistry of human CCA tissues revealed increased PRKAR1A with decreased PRKAR2B protein expression. Moreover, CCA cell lines showed abundantly expressed PRKAR1A, while lacking PRKAR2B expression. Silencing PRKAR1A expression induced growth inhibition and apoptosis of CCA cells, with an associated decrease in mitogen-activated protein kinases, PI3K/Akt, JAK/STAT and Wnt/β-catenin pathway signaling. The inhibition of PKA using a PKA inhibitor and cAMP analogs also led to a significant cell growth inhibition. In conclusion, our study reports the overexpression as well as molecular mechanisms by which PRKAR1A/PKA regulates human CCA cell growth. Importantly, abrogation of gene expression caused significant CCA cell growth inhibition, oncogenic signaling and coupled apoptosis induction, suggesting PRKAR1A's potential as a drug target for CCA therapy., (Copyright © 2010 UICC.)

Human T-cell leukemia virus type 1 (HTLV-1) is the etiologic agent for adult T-cell leukemia (ATL). Aurora A, a mitotic checkpoint protein, is overexpressed in human cancer cells. The cell cycle-dependent turnover of Aurora A is regulated by E3 ubiquitin ligases such as checkpoint with fork head-associated and ring finger (CHFR). Here, we found overexpression of Aurora A protein in HTLV-1-infected T-cell lines and primary ATL cells. The expression of CHFR mRNA was reduced in these cells by abnormal methylation of CHFR promoter region. Knockdown of Aurora A using small interfering RNA suppressed the growth of HTLV-1-infected T-cell line. Transfection of Aurora A expression plasmid enhanced Tax-induced nuclear factor-kappaB (NF-kappaB) reporter activity. Transfection of CHFR expression plasmid into an HTLV-1-infected T-cell line reduced cell growth, Aurora A protein level and constitutive NF-kappaB reporter activity. Aurora kinase inhibitor suppressed the growth and survival of HTLV-1-infected T-cell lines and primary ATL cells. It also reduced constitutive NF-kappaB activity in an HTLV-1-infected T-cell line by reducing IkappaB kinase beta phosphorylation and the expression of antiapoptotic protein survivin. Our results suggested that loss of CHFR expression resulted to accumulation of Aurora A, which increased NF-kappaB activity. These findings highlight the critical role of Aurora A in HTLV-1-infected T cells, making this molecule a potentially suitable target for future therapies for ATL.

p53 protein is a transcription factor involved in multiple tumor-suppressor activities including cell cycle control and apoptosis. TP53 gene is frequently mutated in glioblastoma, suggesting the importance of inactivation of this gene product in gliomagenesis. Restoration of p53 function in glioblastoma cell lines deficient for p53 has shown that p53 induces growth arrest or apoptosis depending on the cell line and vector used to transduce wild-type TP53 alleles. Considering that astrocytes grow and express p53, it is not clear whether these results reflect physiologic responses or the result of p53 overexpression in combination with cellular responses to viral vector infection. Here, we reassessed this issue using a glioblastoma cell line (LN382) that expresses an endogenous temperature-sensitive mutant p53. This cell line expresses TP53 alleles (100% as determined by a p53 transcriptional assay in yeast) mutated at codon 197 GTG (Val) > CTG (Leu). We found that the p53 protein in these cells acted as an inactive mutant at 37 degrees C and as a functional wild-type p53 below 34 degrees C as demonstrated by several lines of evidence, including (i) restoration of transactivating ability in yeast, (ii) induction of p53-modulated genes such as CDKN1(p21) and transforming growth factor-alpha, (iii) disappearance of accumulated p53 protein in the nucleus and (iv) decrease in steady state p53 protein levels. This temperature switch allowed p53 levels, which were close to physiological levels to dramatically reduce LN382 cell proliferation by inducing a G(1)/S cell cycle block, but not to induce apoptosis. The lack of apoptosis was considered to be a result of the low level p53 expression, because increasing wild-type p53 levels by adenoviral-mediated gene transfer caused apoptosis in these cells. The LN382 cell line will be extremely useful for investigations into the roles of p53 in cellular responses to a variety of stimuli or damages., (Copyright 2001 Wiley-Liss, Inc.)

Membrane-associated guanylate kinases (MAGUKs) are known to function as scaffolds for forming multiprotein complexes at the synaptic junctions of neuronal cells and at sites of epithelial cell-cell contact. In Drosophila, mutations of the lethal (1)-discs large (dlg) gene, which encodes a MAGUK protein, leads to post-synaptic structure defects in neuronal cells and neoplastic overgrowth of epithelial cells. We previously showed that NE-dlg (neuronal and endocrine dlg), a human homolog of the dlg, plays a crucial role in formation of synaptic structure in human neuronal cells. Here we demonstrate that NE-dlg, similar to Drosophila dlg, is involved in regulation of cell cycle progression and adhesive ability of non-neuronal cells. Overexpression of NE-dlg in proliferating cells including various cancer cell lines induced growth suppression and impairment of cell adhesive ability. Furthermore, NE-dlg overexpression caused the down-regulation of beta-catenin in cancer cells regardless of mutations in the APC (adenomatous polyposis coli) gene. The PDZ domains of NE-dlg were found to be essential for the growth suppression, loss of adhesive property and down-regulation of beta-catenin. We propose that NE-dlg regulates the cell growth and adhesive ability by controlling the level of beta-catenin through an APC-independent pathway. Inactivation of NE-dlg may therefore contribute to development and/or progression of human neoplasms., (Copyright 2000 Wiley-Liss, Inc.)

We examined the frequency of p53 mutations in 38 oropharyngeal squamous cell carcinomas (SCC), using both a yeast functional assay and a conventional immunohistochemical staining method (IHC) to detect p53 mutations. We also explored the clinical importance of p53 mutations in oropharyngeal SCC. An accumulation of p53 protein was detected in 17 of the 38 (45%) tumors by IHC, whereas the yeast-based assay detected 6 additional p53 mutations, for a total of 23 tumors (61%) with p53 mutations. The cDNA sequencing analysis revealed that the 6 mutations undetected by IHC consisted of 3 frameshift, 1 nonsense and 2 missense mutations. Thus, the yeast functional assay was more sensitive than conventional IHC for detecting p53 mutations. Subsequently, the relationship between p53 mutations and the clinico-pathological parameters in oropharyngeal SCC was evaluated using the results of the functional assay. Mutation of p53 was not associated with the patient age, sex, tumor stage or degree of tumor cell differentiation. Interestingly, heavy drinking had a significant positive correlation with the p53 mutation, but heavy smoking did not, suggesting that prolonged exposure to alcohol is more related to p53 mutation in oropharyngeal SCC than to tobacco consumption. Radiation sensitivity was examined by comparing tumor size on magnetic resonance images before and after completion of therapy with 45 Gy radiation, in the 18 cases of T2 oropharyngeal SCC that were initially treated by radiotherapy. The results showed that tumors with wild-type p53 decreased in size significantly compared to those with mutant p53. In 33 patients treated with curative intent, the overall survival after the completion of therapy was better in patients with a wild-type p53 tumor than in patients with a mutant p53 tumor. We conclude that p53 mutation is associated with radiation resistance and a decreased probability of survival in oropharyngeal SCC., (Copyright 2000 Wiley-Liss, Inc.)

CD44 is a widely expressed cell surface adhesion molecule in which various isoforms arise from alternative RNA splicing mechanism. Overexpression of specific CD44 splice variant, i.e., CD44v8-10, has been found in several human malignancies and is considered to be associated with tumor progression and metastasis. We have demonstrated a novel molecular approach, CD44v8-10/CD44v10 competitive reverse transcription-polymerase chain reaction (CC-RT-PCR) for the detection of cancer cells overexpressing CD44v8-10 by the measurement of the transcriptional level of CD44v8-10 relative to that of CD44v10 (v8-10/v10 ratio). In this study, we initially examined the expression of CD44 splice variants in human urothelial cancers and their adjacent normal urinary tissues by RT-PCR. Any CD44 variant isoforms were barely detectable in normal urinary tissues, whereas CD44v8-10 was predominantly expressed in 23 of the 30 (77%) urothelial cancer specimens. We then applied CC-RT-PCR to spontaneously voided urine samples from patients with 80 urothelial cancer and 50 various benign urologic diseases. The CC-RT-PCR analysis revealed that all of the samples associated with benign diseases presented a predominant expression of the CD44v10 transcript (the v8-10/v10 ratios = 0.00-0.87), whereas 62 of the 80 samples associated with urothelial cancers mostly expressed the CD44v8-10 transcript (the v8-10/v10 ratios > 1.00). In addition, the positivity rate obtained by the CC-RT-PCR analysis was high regardless of the pathological grade of the urothelial cancers, although the sensitivity of the cytological examination declined with decreasing tumor grade. Our findings suggest strongly that CC-RT-PCR is a non-invasive useful tool for the diagnosis of urine samples from patients with urothelial cancer.

Identification of genetic structure and diversity of T-cell receptor (TCR)alpha and beta genes for cytotoxic T lymphocytes (CTLs) infiltrating human cancers is important for the better understanding of molecular mechanisms of host defense at tumor sites. cDNAs of TCR alpha and beta genes of 22 different melanoma-specific CTL clones established from the tumor-infiltrating lymphocytes of 2 patients were sequenced for analysis of their genetic structure and diversity. V alpha 7.2-J alpha 10-C alpha was found in 4 of 22 clones, 2 of which also used the same beta-chain. The other 20 clones showed different combinations of alpha and beta use. At deduced amino-acid levels, 7 of 9 clones from one patient used a threonine residue at the 26th position in the complementarity-determining region (CDR)1 of TCR alpha. Eight of 13 clones used a threonine at the 99th or a serine residue at the 100th position in CDR3 of TCR alpha CTL clones with the same or different TCR alpha showed the same or different patterns of cytotoxicity, respectively. These results suggest that CTLs usually do not demonstrate clonal expansion at tumor sites of metastatic melanoma's but rather that polyclonal T cells capable of binding to multiple melanoma determinants through CDR3 of TCR alpha accumulate in the tumor.

The present study was undertaken to determine the role of the metalloproteinase MMP-9 in the invasive phenotype of squamous-cell carcinoma of the oral cavity and the regulation of its expression. Zymographic analysis of conditioned medium from 2 highly invasive squamous-cell-carcinoma cell lines indicated large amounts of an enzyme which was indistinguishable, in size (92 kDa) from the MMP-9 pro-enzyme. Conversion of the 92-kDa gelatinase into a lower-molecular-weight species (84 kDa), identical in size to the activated gelatinase, was evident when both cell lines, which are avid secretors of urokinase, were cultured in the presence of plasminogen. Penetration of an extracellular-matrix-coated filter was dramatically reduced in the presence of the collagenase inhibitor, tissue inhibitor of metalloproteinase-2, suggesting a critical role for MMP-9 in the invasive process. Immunohistochemical studies demonstrating the presence of MMP-9 in tumor cells of resected squamous-cell cancers suggested that secretion of this collagenase by cells in vitro was reflective of the in vivo setting. Since several phorbol-ester response elements are present in the MMP-9 promoter, we determined the role of protein-kinase-C pathways in the regulation of MMP-9 expression in cultured SCC. Treatment of cells with PMA resulted in a more-than-20-fold increase in the level of protein and mRNA. Conversely, culturing of cells in the presence of the protein-kinase-C inhibitor, calphostin-C, led to a dose-dependent decrease in the amount of MMP-9 mRNA and protein, suggesting that the constitutive expression of this collagenase reflects activation of this signal transduction pathway. In summary, our data suggest that, for a sub-population of squamous-cell carcinomas, secreted MMP-9 is an important determinant of the invasive phenotype, and that the expression of this metalloproteinase is regulated by protein-kinase-C pathways.

A rat monoclonal antibody (MAb) termed RS-II (Ig M) was obtained by syngeneic immunization with rat bladder tumor cells induced by N-butyl, N-hydroxybutylnitrosamine (BBN). Immunocytochemical analysis showed that RS-II is also reactive with mouse, dog and human bladder tumor-cell lines and some other human tumor-cell lines but not myeloma or leukemia cells. Immunohistochemical examination of paraffin-embedded tissues has shown that RS-II is reactive with mouse, rat, dog and human bladder tumors (5/5, 5/5, 1/1, 31/49) and some other tumors, but not with normal human urothelium or normal rat tissues. The antigen is expressed on the majority of low-grade or well-differentiated tumors, but less on advanced, invasive tumors. The immunofluorescence staining pattern of cultured cells was cytoplasmic and filamentous. Immunoblotting revealed that the antigen is a Mr 54,000 to Mr 56,000 protein which was extracted with difficulty from the cultured cells with Triton X-100. The antigen was also detected in the culture supernatant by means of ELISA. Our results suggest that the epitope is expressed on cytoskeletons common to a wide variety of mammalian cells at a certain stage of differentiation.