Emodin, a tyrosine kinase inhibitor, and human cancer

Many human diseases, including cancers, result from aberrations of signal transduction pathways. The recent understanding of the molecular biochemistry of signal transduction in normal and transformed cells enable us to have a better insight about cancer and design new drugs to target this abnormal signaling in the cancer cells. Tyrosine kinase pathway plays a very important role in normal and cancer cells. Enhanced activity of tyrosine kinases has been associated with many human cancer types. Therefore, identifying the type of tyrosine kinases involved in a particular cancer type and blocking these tyrosine kinase pathways may provide a way to treat cancer. Receptor tyrosine kinase expression, namely epidermal growth factor receptor (EGFR) family, was examined in the oral squamous cell carcinoma patients. The expression levels of different members of the EGFR family were found to be significantly associated with shorter patients' survival. Combining EGFR, HER-2/neu, and HER-3 expression can significantly improve the predicting power. The effect of emodin, a tyrosine kinase inhibitor, on these receptors in head and neck squamous cell carcinoma cell lines was examined. Emodin was found to suppress the tyrosine phosphorylation of HER-2/neu and EGF-induced tyrosine phosphorylation of EGFR. Emodin also induced apoptosis and downregulated the expression of anti-apoptotic protein bcl-2 in oral squamous cell carcinoma cells. It is known that tyrosine kinase pathways are involved in estrogen receptor signaling pathway. Therefore, the effects of inhibiting the tyrosine kinase pathway in estrogen receptor-positive breast cancers was studied. Emodin was found to act similarly to antiestrogens, capable of inhibiting estrogen-stimulated growth and DNA synthesis, and the phosphorylation of Rb protein. Interestingly, emodin, and other tyrosine kinase inhibitors, such as RG 13022 and genistein, depleted cellular levels of estrogen receptor protein. Emodin-induced depletion of estrogen receptor was mediated by the proteasome degradation pathway. In summary, we have demonstrated that tyrosine kinase pathways play an important role in oral squamous cell carcinoma and estrogen receptor-positive breast cancer. Targeting the tyrosine kinases by inhibitors, such as emodin, may provide a potential way to treat the cancer patients. ^

RNA polymerase II large subunit degradation induced by ecteinascidin 743: Molecular characterization and subsequent rational investigation of antitumor mechanisms in cancers with clinical response

Ecteinascidin 743 (Et-743), which is a novel DNA minor groove alkylator with a unique spectrum of antitumor activity, is currently being evaluated in phase II/III clinical trials. Although the precise molecular mechanisms responsible for the observed antitumor activity are poorly understood, recent data suggests that post-translational modifications of RNA polymerase II Large Subunit (RNAPII LS) may play a central role in the cellular response to this promising anticancer agent. The stalling of an actively transcribing RNAPII LS at Et-743-DNA adducts is the initial cellular signal for transcription-coupled nucleotide excision repair (TC-NER). In this manner, Et-743 poisons TC-NER and produces DNA single strand breaks. Et-743 also inhibits the transcription and RNAPII LS-mediated expression of selected genes. Because the poisoning of TC-NER and transcription inhibition are critical components of the molecular response to Et-743 treatment, we have investigated if changes in RNAPII LS contribute to the disruption of these two cellular pathways. In addition, we have studied changes in RNAPII LS in two tumors for which clinical responses were reported in phase I/II clinical trials: renal cell carcinoma and Ewing's sarcoma. Our results demonstrate that Et-743 induces degradation of the RNAPII LS that is dependent on active transcription, a functional 26S proteasome, and requires functional TC-NER, but not global genome repair. Additionally, we have provided the first experimental data indicating that degradation of RNAPII LS might lead to the inhibition of activated gene transcription. A set of studies performed in isogenic renal carcinoma cells deficient in von Hippel-Lindau protein, which is a ubiquitin-E3-ligase for RNAPII LS, confirmed the central role of RNAPII LS degradation in the sensitivity to Et-743. Finally, we have shown that RNAPII LS is also degraded in Ewing's sarcoma tumors following Et-743 treatment and provide data to suggest that this event plays a role in decreased expression of the Ewing's sarcoma oncoprotein, EWS-Fli1. Altogether, these data implicate degradation of RNAPII LS as a critical event following Et-743 exposure and suggest that the clinical activity observed in renal carcinoma and Ewing's sarcoma may be mediated by disruption of molecular pathways requiring a fully functional RNAPII LS. ^

Non-p53 p53RE binding protein, a human transcription factor functionally analogous to P53

The transactivation activity of the p53 tumor suppressor protein is critical for regulating cell growth and apoptosis. We describe the identification of a transcription factor that is functionally similar to p53 and contains the same DNA binding and transcription activities specific for the p53 responsive DNA element (p53RE). This protein was highly purified through chromatography from HeLa cell extracts. The purified protein was able to bind specifically to the p53RE derived from a p21waf1 promoter and to stimulate p53RE-dependent transcription but not basal transcription in vitro. Its DNA-binding activity was inhibited by the wild type but not mutant p53RE-containing DNA oligomers. Also, this p53RE-binding activity was found in human p53 null Saos-2 osteosarcoma and H1299 small cell lung carcinoma cells. Interestingly, this activity exhibited a p53RE sequence preference that was distinct from the p53 protein. The activity is neither p53 nor p73, because anti-p53 or anti-73 antibodies were unable to detect this purified protein nor were the antibodies able to alter the p53-like activity, the p53RE-protein complex. These results demonstrate that, besides p73, an additional p53-like protein exists in cells, which is named NBP for non-p53, p53RE binding protein.

Photodynamic therapy results in induction of WAF1/CIP1/P21 leading to cell cycle arrest and apoptosis

Photodynamic therapy (PDT) is a promising new modality that utilizes a combination of a photosensitizing chemical and visible light for the management of a variety of solid malignancies. The mechanism of PDT-mediated cell killing is not well defined. We investigated the involvement of cell cycle regulatory events during silicon phthalocyanine (Pc4)-PDT-mediated apoptosis in human epidermoid carcinoma cells A431. PDT resulted in apoptosis, inhibition of cell growth, and G0-G1 phase arrest of the cell cycle, in a time-dependent fashion. Western blot analysis revealed that PDT results in an induction of the cyclin kinase inhibitor WAF1/CIP1/p21, and a down-regulation of cyclin D1 and cyclin E, and their catalytic subunits cyclin-dependent kinase (cdk) 2 and cdk6. The treatment also resulted in a decrease in kinase activities associated with all the cdks and cyclins examined. PDT also resulted in (i) an increase in the binding of cyclin D1 and cdk6 toward WAF1/CIP1/p21, and (ii) a decrease in the binding of cyclin D1 toward cdk2 and cdk6. The binding of cyclin E and cdk2 toward WAF1/CIP1/p21, and of cyclin E toward cdk2 did not change by the treatment. These data suggest that PDT-mediated induction of WAF1/CIP1/p21 results in an imposition of artificial checkpoint at G1 → S transition thereby resulting in an arrest of cells in G0-G1 phase of the cell cycle through inhibition in the cdk2, cdk6, cyclin D1, and cyclin E. We suggest that this arrest is an irreversible process and the cells, unable to repair the damages, ultimately undergo apoptosis.

Abrogation of the G2 cell cycle checkpoint associated with overexpression of HSIX1: A possible mechanism of breast carcinogenesis

While conducting a search for cell cycle-regulated genes in human mammary carcinoma cells, we identified HSIX1, a recently discovered member of a new homeobox gene subfamily. HSIX1 expression was absent at the onset of and increased toward the end of S phase. Since its expression pattern is suggestive of a role after S phase, we investigated the effect of HSIX1 in the G2 cell cycle checkpoint. Overexpression of HSIX1 in MCF7 cells abrogated the G2 cell cycle checkpoint in response to x-ray irradiation. HSIX1 expression was absent or very low in normal mammary tissue, but was high in 44% of primary breast cancers and 90% of metastatic lesions. In addition, HSIX1 was expressed in a variety of cancer cell lines, suggesting an important function in multiple tumor types. These data support the role for homeobox genes in tumorigenesis/tumor progression, possibly through a cell cycle function.

Two-component kinase-like activity of nm23 correlates with its motility-suppressing activity

Nm23 genes, which encode nucleoside diphosphate kinases, have been implicated in suppressing tumor metastasis. The motility of human breast carcinoma cells can be suppressed by transfection with wild-type nm23-H1, but not by transfections with two nm23-H1 mutants, nm23-H1S12OG and nm23-H1P96S. Here we report that nm23-H1 can transfer a phosphate from its catalytic histidine to aspartate or glutamate residues on 43-kDa membrane proteins. One of the 43-kDa membrane proteins was not phosphorylated by either nm23-H1P96S or nm23-H1S120G, and another was phosphorylated much more slowly by nm23-H1P96S and by nm23-H1S120G than by wild-type nm23-H1. Nm23-H1 also can transfer phosphate from its catalytic histidine to histidines on ATP-citrate lyase and succinic thiokinase. The rates of phosphorylation of ATP-citrate lyase by nm23-H1S120G and nm23-H1P96S were similar to that by wild-type nm23-H1. The rate of phosphorylation of succinic thiokinase by nm23-H1S120 was similar to that by wild-type nm23-H1, and the rate of phosphorylation of succinic thiokinase by nm23-H1P96S was about half that by wild-type nm23-H1. Thus, the transfer of phosphate from nm23-H1 to aspartates or glutamates on other proteins appears to correlate better with the suppression of motility than does the transfer to histidines.

Hypo-phosphorylation of the retinoblastoma protein (pRb) by cyclin D:Cdk4/6 complexes results in active pRb

In cycling cells, the retinoblastoma protein (pRb) is un- and/or hypo-phosphorylated in early G1 and becomes hyper-phosphorylated in late G1. The role of hypo-phosphorylation and identity of the relevant kinase(s) remains unknown. We show here that hypo-phosphorylated pRb associates with E2F in vivo and is therefore active. Increasing the intracellular concentration of the Cdk4/6 specific inhibitor p15INK4b by transforming growth factor β treatment of keratinocytes results in G1 arrest and loss of hypo-phosphorylated pRb with an increase in unphosphorylated pRb. Conversely, p15INK4b-independent transforming growth factor β-mediated G1 arrest of hepatocellular carcinoma cells results in loss of Cdk2 kinase activity with continued Cdk6 kinase activity and pRb remains only hypo-phosphorylated. Introduction of the Cdk4/6 inhibitor p16INK4a protein into cells by fusion to a protein transduction domain also prevents pRb hypo-phosphorylation with an increase in unphosphorylated pRb. We conclude that cyclin D:Cdk4/6 complexes hypo-phosphorylate pRb in early G1 allowing continued E2F binding.

The mechanism of cancer-mediated conversion of plasminogen to the angiogenesis inhibitor angiostatin

Angiostatin, a potent naturally occurring inhibitor of angiogenesis and growth of tumor metastases, is generated by cancer-mediated proteolysis of plasminogen. Human prostate carcinoma cells (PC-3) release enzymatic activity that converts plasminogen to angiostatin. We have now identified two components released by PC-3 cells, urokinase (uPA) and free sulfhydryl donors (FSDs), that are sufficient for angiostatin generation. Furthermore, in a defined cell-free system, plasminogen activators [uPA, tissue-type plasminogen activator (tPA), or streptokinase], in combination with one of a series of FSDs (N-acetyl-l-cysteine, d-penicillamine, captopril, l-cysteine, or reduced glutathione] generate angiostatin from plasminogen. An essential role of plasmin catalytic activity for angiostatin generation was identified by using recombinant mutant plasminogens as substrates. The wild-type recombinant plasminogen was converted to angiostatin in the setting of uPA/FSD; however, a plasminogen activation site mutant and a catalytically inactive mutant failed to generate angiostatin. Cell-free derived angiostatin inhibited angiogenesis in vitro and in vivo and suppressed the growth of Lewis lung carcinoma metastases. These findings define a direct mechanism for cancer-cell-mediated angiostatin generation and permit large-scale production of bioactive angiostatin for investigation and potential therapeutic application.

p53-dependent regulation of MDR1 gene expression causes selective resistance to chemotherapeutic agents

Loss of functional p53 paradoxically results in either increased or decreased resistance to chemotherapeutic drugs. The inconsistent relationship between p53 status and drug sensitivity may reflect p53’s selective regulation of genes important to cytotoxic response of chemotherapeutic agents. We reasoned that the discrepant effects of p53 on chemotherapeutic cytotoxicity is due to p53-dependent regulation of the multidrug resistance gene (MDR1) expression in tumors that normally express MDR1. To test the hypothesis that wild-type p53 regulates the endogenous mdr1 gene we stably introduced a trans-dominant negative (TDN) p53 into rodent H35 hepatoma cells that express P-glycoprotein (Pgp) and have wild-type p53. Levels of Pgp and mdr1a mRNA were markedly elevated in cells expressing TDN p53 and were linked to impaired p53 function (both transactivation and transrepression) in these cells. Enhanced mdr1a gene expression in the TDN p53 cells was not secondary to mdr1 gene amplification and Pgp was functional as demonstrated by the decreased uptake of vinblastine. Cytotoxicity assays revealed that the TDN p53 cell lines were selectively insensitive to Pgp substrates. Sensitivity was restored by the Pgp inhibitor reserpine, demonstrating that only drug retention was the basis for loss of drug sensitivity. Similar findings were evident in human LS180 colon carcinoma cells engineered to overexpress TDN p53. Therefore, the p53 inactivation seen in cancers likely leads to selective resistance to chemotherapeutic agents because of up-regulation of MDR1 expression.

Potent inhibition of tumor survival in vivo by β-lapachone plus taxol: Combining drugs imposes different artificial checkpoints

Ablation of tumor colonies was seen in a wide spectrum of human carcinoma cells in culture after treatment with the combination of β-lapachone and taxol, two low molecular mass compounds. They synergistically induced death of cultured ovarian, breast, prostate, melanoma, lung, colon, and pancreatic cancer cells. This synergism is schedule dependent; namely, taxol must be added either simultaneously or after β-lapachone. This combination therapy has unusually potent antitumor activity against human ovarian and prostate tumor prexenografted in mice. There is little host toxicity. Cells can commit to apoptosis at cell-cycle checkpoints, a mechanism that eliminates defective cells to ensure the integrity of the genome. We hypothesize that when cells are treated simultaneously with drugs activating more than one different cell-cycle checkpoint, the production of conflicting regulatory signaling molecules induces apoptosis in cancer cells. β-Lapachone causes cell-cycle delays in late G1 and S phase, and taxol arrests cells at G2/M. Cells treated with both drugs were delayed at multiple checkpoints before committing to apoptosis. Our findings suggest an avenue for developing anticancer therapy by exploiting apoptosis-prone “collisions” at cell-cycle checkpoints.

The R1 component of mammalian ribonucleotide reductase has malignancy-suppressing activity as demonstrated by gene transfer experiments

Our recent studies have shown that deregulated expression of R2, the rate-limiting component of ribonucleotide reductase, enhances transformation and malignant potential by cooperating with activated oncogenes. We now demonstrate that the R1 component of ribonucleotide reductase has tumor-suppressing activity. Stable expression of a biologically active ectopic R1 in ras-transformed mouse fibroblast 10T½ cell lines, with or without R2 overexpression, led to significantly reduced colony-forming efficiency in soft agar. The decreased anchorage independence was accompanied by markedly suppressed malignant potential in vivo. In three ras-transformed cell lines, R1 overexpression resulted in abrogation or marked suppression of tumorigenicity. In addition, the ability to form lung metastases by cells overexpressing R1 was reduced by >85%. Metastasis suppressing activity also was observed in the highly malignant mouse 10T½ derived RMP-6 cell line, which was transformed by a combination of oncogenic ras, myc, and mutant p53. Furthermore, in support of the above observations with the R1 overexpressing cells, NIH 3T3 cells cotransfected with an R1 antisense sequence and oncogenic ras showed significantly increased anchorage independence as compared with control ras-transfected cells. Finally, characteristics of reduced malignant potential also were demonstrated with R1 overexpressing human colon carcinoma cells. Taken together, these results indicate that the two components of ribonucleotide reductase both are unique malignancy determinants playing opposing roles in its regulation, that there is a novel control point important in mechanisms of malignancy, which involves a balance in the levels of R1 and R2 expression, and that alterations in this balance can significantly modify transformation, tumorigenicity, and metastatic potential.

SMAD3/4-dependent transcriptional activation of the human type VII collagen gene (COL7A1) promoter by transforming growth factor β

The human type VII collagen gene (COL7A1) recently has been identified as an immediate-early response gene for transforming growth factor β (TGF-β)/SMAD signaling pathway. In this study, by using MDA-MB-468 SMAD4−/− breast carcinoma cells, we demonstrate that expression of SMAD4 is an absolute requirement for SMAD-mediated promoter activity. We also demonstrate that the SMAD binding sequence (SBS) representing the TGF-β response element in the region −496/−444 of the COL7A1 promoter functions as an enhancer in the context of a heterologous promoter. Electrophoretic mobility-shift assays with nuclear extracts from COS-1 cells transfected with expression vectors for SMADs 1–5 indicate that SMAD3 forms a complex with a migration similar to that of the endogenous TGF-β-specific complex observed in fibroblast extracts. Electrophoretic mobility-shift assays using recombinant glutathione S-transferase-SMAD fusion proteins indicate that both SMAD4 and C-terminally truncated SMAD3, but not SMAD2, can bind the COL7A1 SBS. Coexpression of SMAD3 and SMAD4 in COS-1 cells leads to the formation of two complexes: a DNA/protein complex containing SMAD3 alone and another slower-migrating complex containing both SMAD3 and SMAD4, the latter complex not being detected in fibroblasts. Maximal transactivation of COL7A1 SBS-driven promoters in either MDA-MB-468 carcinoma cells or fibroblasts requires concomitant overexpression of SMAD3 and SMAD4. These data may represent the first identification of a functional homomeric SMAD3 complex regulating a human gene.

Thrombospondin-2: A potent endogenous inhibitor of tumor growth and angiogenesis

Recent evidence suggests a potential role for thrombospondin-2 (TSP-2), a matricellular glycoprotein, in the regulation of primary angiogenesis. To directly examine the biological effect of TSP-2 expression on tumor growth and angiogenesis, human A431 squamous cell carcinoma cells, which do not express TSP-2, were stably transfected with a murine TSP-2 expression vector or with vector alone. A431 cells expressing TSP-2 did not show an altered growth rate, colony-forming ability, or susceptibility to induction of apoptosis in vitro. However, injection of TSP-2-transfected clones into the dermis of nude mice resulted in pronounced inhibition of tumor growth that was significantly stronger than the inhibition observed in A431 clones stably transfected with a thrombospondin-1 (TSP-1) expression vector, and combined overexpression of TSP-1 and TSP-2 completely prevented tumor formation. Extensive areas of necrosis were observed in TSP-2-expressing tumors, and both the density and the size of tumor vessels were significantly reduced, although tumor cell expression of the major tumor angiogenesis factor, vascular endothelial growth factor, was maintained at high levels. These findings establish TSP-2 as a potent endogenous inhibitor of tumor growth and angiogenesis.

Transforming growth factor β targeted inactivation of cyclin E:cyclin-dependent kinase 2 (Cdk2) complexes by inhibition of Cdk2 activating kinase activity

Transforming growth factor β (TGF-β)-mediated G1 arrest previously has been shown to specifically target inactivation of cyclin D:cyclin-dependent kinase (Cdk) 4/6 complexes. We report here that TGF-β-treated human HepG2 hepatocellular carcinoma cells arrest in G1, but retain continued cyclin D:Cdk4/6 activity and active, hypophosphorylated retinoblastoma tumor suppressor protein. Consistent with this observation, TGF-β-treated cells failed to induce p15INK4b, down-regulate CDC25A, or increase levels of p21CIP1, p27KIP1, and p57KIP2. However, TGF-β treatment resulted in the specific inactivation of cyclin E:Cdk2 complexes caused by absence of the activating Thr160 phosphorylation on Cdk2. Whole-cell lysates from TGF-β-treated cells showed inhibition of Cdk2 Thr160 Cdk activating kinase (CAK) activity; however, cyclin H:Cdk7 activity, a previously assumed mammalian CAK, was not altered. Saccharomyces cerevisiae contains a genetically and biochemically proven CAK gene, CAK1, that encodes a monomeric 44-kDa Cak1p protein unrelated to Cdk7. Anti-Cak1p antibodies cross-reacted with a 45-kDa human protein with CAK activity that was specifically down-regulated in response to TGF-β treatment. Taken together, these observations demonstrate that TGF-β signaling mediates a G1 arrest in HepG2 cells by targeting Cdk2 CAK and suggests the presence of at least two mammalian CAKs: one specific for Cdk2 and one for Cdk4/6.

Expression cloning of cDNA encoding a human β-1,3-N-acetylglucosaminyltransferase that is essential for poly-N-acetyllactosamine synthesis

The structure and biosynthesis of poly-N-acetyllactosamine display a dramatic change during development and oncogenesis. Poly-N-acetyllactosamines are also modified by various carbohydrate residues, forming functional oligosaccharides such as sialyl Lex. Herein we describe the isolation and functional expression of a cDNA encoding β-1,3-N-acetylglucosaminyltransferase (iGnT), an enzyme that is essential for the formation of poly-N-acetyllactosamine. For this expression cloning, Burkitt lymphoma Namalwa KJM-1 cells were transfected with cDNA libraries derived from human melanoma and colon carcinoma cells. Transfected Namalwa cells overexpressing the i antigen were continuously selected by fluorescence-activated cell sorting because introduced plasmids containing Epstein–Barr virus replication origin can be continuously amplified as episomes. Sibling selection of plasmids recovered after the third consecutive sorting resulted in a cDNA clone that directs the increased expression of i antigen on the cell surface. The deduced amino acid sequence indicates that this protein has a type II membrane protein topology found in almost all mammalian glycosyltransferases cloned to date. iGnT, however, differs in having the longest transmembrane domain among glycosyltransferases cloned so far. The iGnT transcript is highly expressed in fetal brain and kidney and adult brain but expressed ubiquitously in various adult tissues. The expression of the presumed catalytic domain as a fusion protein with the IgG binding domain of protein A enabled us to demonstrate that the cDNA encodes iGnT, the enzyme responsible for the formation of GlcNAcβ1 → 3Galβ1 → 4GlcNAc → R structure and poly-N-acetyllactosamine extension.