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Altered expression of Urea Cycle (UC) enzymes occurs in many tumors, resulting a metabolic hallmark termed as UC dysregulation. Polyamines are synthesized from ornithine, and polyamine synthetic genes are elevated in various tumors. However, the underlying deregulations of UC/ polyamine synthesis in cancer remain elusive. Here, we characterized a hypoxia-induced lncRNA LVBU (lncRNA regulation via BCL6/urea cycle) that is highly expressed in colorectal cancer (CRC) and correlates with poor cancer prognosis. Increased LVBU expression promoted CRC cells proliferation, foci formation and tumorigenesis. Further, LVBU regulates urea cycle and polyamine synthesis through BCL6, a negative regulator of p53. Mechanistically, overexpression of LVBU competitively bound miR-10a/miR-34c to protect BCL6 from miR-10a/34c-mediated degradation, which in turn allows BCL6 to block p53-mediated suppression of genes (arginase1 ARG1, ornithine transcarbamylase OTC, ornithine decarboxylase 1 ODC1) involved in UC/polyamine synthesis. Significantly, ODC1 inhibitor attenuated the growth of patient derived xenografts (PDX) that sustain high LVBU levels. Taken together, elevated LVBU can regulate BCL6-p53 signaling axis for systemic UC/polyamine synthesis reprogramming and confers a predilection toward CRC development. Our data demonstrates that further drug development and clinical evaluation of inhibiting UC/polyamine synthesis are warranted for CRC patients with high expression of LVBU.

Colon cancer is one of the most common cancers with high mortality in humans. Early diagnosis and treatment of colon cancer is of great significance for cancer therapy. Numerous theranostic agents have been developed to detect and kill cancer cells. However, few reports have focused on how these agents control and affect the gene expression of cancer cells in vivo. Herein, three pyridinium-functionalized tetraphenylethylene derivatives, namely, TPE-OM, TPE-H, and TPE-NO2, with electron-donating and electron-withdrawing groups were facilely synthesized as theranostic agents for cell imaging and anticolon cancer therapy. Among these AIE luminogens (AIEgens), TPE-OM with donor and acceptor structure showed the best treatment efficacy for colon cancer through systematic biological evaluation and comparison. Both in vitro cell imaging and in vivo tumor treatment experiments demonstrated that TPE-OM can be utilized as an efficient theranostic agent to diagnose and kill colon cancer cells. Flow cytometric analysis revealed that the cell cycle process was disturbed by TPE-OM in colon cancer cells. Deep insight into the gene level revealed that the expressions of cell-cycle-promoting genes was inhibited upon addition of TPE-OM. This study may open a new venue for unraveling the mechanisms of cancer metastasis.

The clinical, epidemiological, and laboratory factors associated with disease severity and mortality from confirmed leptospirosis patients in Taiwan are not well known. This retrospective study examined patients with suspected leptospirosis admitted to En-Chu-Kong Hospital, a regional teaching hospital in northern Taiwan.

The Serine–Glycine–One-Carbon (SGOC) pathway is pivotal in multiple anabolic processes. Expression levels of SGOC genes are deregulated under tumorigenic conditions, suggesting participation of oncogenes in deregulating the SGOC biosynthetic pathway. However, the underlying mechanism remains elusive. Here, we identified that Interleukin enhancer-binding factor 3 (ILF3) is overexpressed in primary CRC patient specimens and correlates with poor prognosis. ILF3 is critical in regulating the SGOC pathway by directly regulating the mRNA stability of SGOC genes, thereby increasing SGOC genes expression and facilitating tumor growth. Mechanistic studies showed that the EGF–MEK–ERK pathway mediates ILF3 phosphorylation, which hinders E3 ligase speckle-type POZ protein (SPOP)-mediated poly-ubiquitination and degradation of ILF3. Significantly, combination of SGOC inhibitor and the anti-EGFR monoclonal antibody cetuximab can hinder the growth of patient-derived xenografts that sustain high ERK-ILF3 levels. Taken together, deregulation of ILF3 via the EGF–ERK signaling plays an important role in systemic serine metabolic reprogramming and confers a predilection toward CRC development. Our findings indicate that clinical evaluation of SGOC inhibitor is warranted for CRC patients with ILF3 overexpression.

ABSTRACTProbiotic roles of Clostridium butyricum(C.B) are involved in regulating disease and cancers, yet the mechanistic basis for these regulatory roles remains largely unknown. Here, we demonstrate that C.B reprograms the proliferation, migration, stemness, and tumor growth in CRC by regulating pivotal signal molecules including MYC. Destabilization of MYC by C.B supplementation suppresses cancer cell proliferation/metastasis, sensitizes 5-FU treatment, and boosts responsiveness of anti-PD1 therapy. MYC is a transcriptional regulator of Thymidylate synthase (TYMS), a key target of the 5-FU. Also MYC is known to impact on PD-1 expression. Mechanistically, C.B treatment of CRC cells results in MYC degradation by enhancing proteasome-mediated ubiquitination, thereby mitigating MYC-mediated 5-FU resistance and boosting anti-PD1 immunotherapeutic efficacy. Together, our findings uncover previously unappreciated links between C.B and CRC cell signaling, providing insight into the tumorigenesis modulating mechanisms of C.B in boosting chemo/immune therapies.

A hypoxic microenvironment contributes to tumor progression, with hypoxia-inducible factor-1α (HIF-1α) being a critical regulator. We have reported that 14-3-3σ is negatively associated with HIF-1α expression; however, its role in hypoxia-induced tumor progression remains poorly characterized. Here we show that 14-3-3σ suppresses cancer hypoxia-induced metastasis and angiogenesis in colorectal cancer (CRC). 14-3-3σ opposes HIF-1α expression by regulating the protein stability of HIF-1α, thereby decreasing HIF-1α transcriptional activity and suppressing tumor progression. Mechanistic studies show that the 14-3-3σ-interacting protein neural precursor cell-expressed developmentally down-regulated 4-like (NEDD4L) is an E3 ligase that targets HIF-1α. 14-3-3σ promotes the binding of S448-phosphorylated NEDD4L to HIF-1α, thereby enhancing HIF-1α poly-ubiquitination and subsequent proteasome-mediated degradation. Consistent with this anti-tumorigenic function for 14-3-3σ, low 14-3-3σ expression levels correlate with poor CRC patient survival, and 14-3-3σ enhances the response of CRC to bevacizumab. These results reveal an important mechanism for 14-3-3σ in tumor suppression through HIF-1α regulation.

The understanding of how hypoxia stabilizes and activates HIF1α in the nucleus with related oncogenic signals could revolutionize targeted therapy for cancers. Here, we find that histone H2AX displays oncogenic activity by serving as a crucial regulator of HIF1α signalling. H2AX interacts with HIF1α to prevent its degradation and nuclear export in order to allow successful VHL-independent HIF1α transcriptional activation. We show that mono-ubiquitylation and phosphorylation of H2AX, which are strictly mediated by hypoxia-induced E3 ligase activity of TRAF6 and ATM, critically regulate HIF1α-driven tumorigenesis. Importantly, TRAF6 and γH2AX are overexpressed in human breast cancer, correlate with activation of HIF1α signalling, and predict metastatic outcome. Thus, TRAF6 and H2AX overexpression and γH2AX-mediated HIF1α enrichment in the nucleus of cancer cells lead to overactivation of HIF1α-driven tumorigenesis, glycolysis and metastasis. Our findings suggest that TRAF6-mediated mono-ubiquitylation and subsequent phosphorylation of H2AX may serve as potential means for cancer diagnosis and therapy.

c-Jun is a proto-oncoprotein that is commonly overexpressed in many types of cancer and is believed to regulate cell proliferation, the cell cycle, and apoptosis by controlling AP-1 activity. Understanding the c-Jun regulation is important to develop treatment strategy for cancer. The COP9 signalosome subunit 6 (CSN6) plays a critical role in ubiquitin-mediated protein degradation. MEKK1 is a serine/threonine kinase and E3 ligase containing PHD/RING domain involved in c-Jun ubiquitination. Here, we show that CSN6 associates with MEKK1 and reduces MEKK1 expression level by facilitating the ubiquitin-mediated degradation of MEKK1. Also we show that CSN6 overexpression diminishes MEKK1-mediated c-Jun ubiquitination, which is manifested in mitigating osmotic stress-mediated c-Jun downregulation. Thus, CSN6 is involved in positively regulating the stability of c-Jun. Overexpression of CSN6 correlates with the upregulation of c-Jun target gene expression in cancer. These findings provide new insight into CSN6-MEKK1-c-Jun axis in tumorigenesis.

The COP9 signalosome subunit 6 (CSN6), which is involved in ubiquitin-mediated protein degradation, is overexpressed in many types of cancer. CSN6 is critical in causing p53 degradation and malignancy, but its target in cell cycle progression is not fully characterized. Constitutive photomorphogenic 1 (COP1) is an E3 ubiquitin ligase associating with COP9 signalosome to regulate important target proteins for cell growth. p27 is a critical G1 CDK inhibitor involved in cell cycle regulation, but its upstream regulators are not fully characterized. Here, we show that the CSN6-COP1 link is regulating p27Kip1stability, and that COP1 is a negative regulator of p27Kip1. Ectopic expression of CSN6 can decrease the expression of p27Kip1, while CSN6 knockdown leads to p27Kip1stabilization. Mechanistic studies show that CSN6 interacts with p27Kip1and facilitates ubiquitin-mediated degradation of p27Kip1. CSN6-mediated p27 degradation depends on the nuclear export of p27Kip1, which is regulated through COP1 nuclear exporting signal. COP1 overexpression leads to the cytoplasmic distribution of p27, thereby accelerating p27 degradation. Importantly, the negative impact of COP1 on p27 stability contributes to elevating expression of genes that are suppressed through p27 mediation. Kaplan-Meier analysis of tumor samples demonstrates that high COP1 expression was associated with poor overall survival. These data suggest that tumors with CSN6/COP1 deregulation may have growth advantage by regulating p27 degradation and subsequent impact on p27 targeted genes.

Breast cancer is a disease of cell cycle, and the dysfunction of cell cycle checkpoints plays a vital role in the occurrence and development of breast cancer. We employed multi-gene fluorescence in situ hybridization (M-FISH) to investigate gene copy number aberrations (CNAs) of 4 genes (Rb1, CHEK2, c-Myc, CCND1) that are involved in the regulation of cell cycle, in order to analyze the impact of gene aberrations on prognosis in the young breast cancer patients. Gene copy number aberrations of these 4 genes were more frequently observed in young breast cancer patients when compared with the older group. Further, these CNAs were more frequently seen in Luminal B type, Her2 overexpression, and tiple-negative breast cancer (TNBC) type in young breast cancer patients. The variations of CCND1, Rb1, and CHEK2 were significantly correlated with poor survival in the young breast cancer patient group, while the amplification of c-Myc was not obviously correlated with poor survival in young breast cancer patients. Thus, gene copy number aberrations (CNAs) of cell cycle-regulated genes can serve as an important tool for prognosis in young breast cancer patients.

HER2/neuoncogene is frequently deregulated in cancers, and the (PI3K)-Akt signaling is one of the major pathways in mediating HER2/neu oncogenic signal. p57Kip2, an inhibitor of cyclin-depependent kinases, is pivotal in regulating cell cycle progression, but its upstream regulators remain unclear. Here we show that the HER2-Akt axis is linked to p57Kip2regulation, and that Akt is a negative regulator of p57Kip2. Ectopic expression of Akt can decrease the expression of p57Kip2, while Akt inhibition leads to p57Kip2stabilization. Mechanistic studies show that Akt interacts with p57Kip2and causes cytoplasmic localization of p57Kip2. Akt phosphorylates p57 on Ser 282 or Thr310. Akt activity results in destabilization of p57 by accelerating turnover rate of p57 and enhancing p57 ubiquitination. Importantly, the negative impact of HER2/Akt on p57 stability contributes to HER2-mediated cell proliferation, transformational activity and tumorigenicity. p57 restoration can attenuate these defects caused by HER2. Significantly, Kaplan-Meier analysis of tumor samples demonstrate that in tumors where HER2 expression was observed, high expression levels of p57Kip2were associated with better overall survival. These data suggest that HER2/Akt is an important negative regulator of p57Kip2, and that p57 restoration in HER2-overexpressing cells can reduce breast tumor growth. Our findings indicate the applicability of employing p57 regulation as a therapeutic intervention in HER2-overexpressing cancers.

Subunit 6 of the COP9 signalosome complex, CSN6, is known to be critical to the regulation of the MDM2-p53 axis for cell proliferation and anti-apoptosis, but its many targets remain unclear. Here we show that p57Kip2is a target of CSN6, and that CSN6 is a negative regulator of p57Kip2. CSN6 associates with p57Kip2, and its overexpression can decrease the steady-state expression of p57Kip2; accordingly, CSN6 deficiency leads to p57Kip2stabilization. Mechanistic studies show that CSN6 associates with p57Kip2and Skp2, a component of the E3 ligase, which, in turn, facilitates Skp2-mediated protein ubiquitination of p57Kip2. Loss of Skp2 compromised CSN6-mediated p57Kip2destabilization, suggesting collaboration between Skp2 and CSN6 in degradation of p57Kip2. CSN6’s negative impact on p57Kip2elevation translates into cell growth promotion, cell cycle deregulation and potentiated transformational activity. Significantly, univariate Kaplan-Meier analysis of tumor samples demonstrates that high CSN6 expression or low p57 expression is associated with poor overall survival. These data suggest that CSN6 is an important negative regulator of p57Kip2, and that overexpression of CSN6 in many types of cancer could lead to decreased expression of p57Kip2and result in promoted cancer cell growth.

HER2/neuoncogene is frequently overexpressed in various types of cancer, and the (PI3K)-Akt signaling pathway is often activated in HER2-overexpressing cancer cells. CSN6, subunit 6 of the COP9 signalosome complex, is pivotal in regulating MDM2 to destabilize p53, but its upstream regulators remain unclear. Here we show that the HER2-Akt axis is linked to CSN6 regulation, and that Akt is a positive regulator of CSN6. Ectopic expression of Akt can increase the expression of CSN6; accordingly, Akt inhibition leads to CSN6 destabilization. Mechanistic studies show that Akt causes CSN6 phosphorylation at Ser 60, which, in turn, reduces ubiquitin-mediated protein degradation of CSN6. Significantly, Akt’s positive impact on CSN6 elevation translates into p53 degradation, potentiating transformational activity and increasing DNA damage. Akt inhibition can attenuate these defects caused by CSN6. These data suggest that Akt is an important positive regulator of CSN6, and that activation of Akt in many types of cancer could lead to abnormal elevation of CSN6 and result in downregulated p53 and increased DNA damage, which promotes cancer cell growth.

FBXW7, a component of E3 ubiquitin ligase, plays an important role in mitotic checkpoint, but its role remains unclear. Aurora B is a mitotic checkpoint kinase that plays a pivotal role in mitosis by ensuring correct chromosome segregation and normal progression through mitosis. Whether Aurora B and FBXW7 are coordinately regulated during mitosis is not known. Here, we show that FBXW7 is a negative regulator for Aurora B. Ectopic expression of FBXW7 can suppress the expression of Aurora B. Accordingly, FBXW7 deficiency leads to Aurora B elevation. Mechanistic studies show that all FBXW7 isoforms are negative regulators of Aurora B expression through ubiquitination-mediated protein degradation. Aurora B interacts with R465 and R505 residues of WD 40 domain of FBXW7. Significantly, inverse correlation between FBXW7 and Aurora B elevation is translated into the deregulation of mitosis. FBWX7 expression mitigates Aurora B-mediated cell growth and mitotic deregulation. In addition, FBXW7 reduces the percentage of multinucleated cells caused by Aurora B overexpression. These data suggest that FBXW7 is an important negative regulator of Aurora B, and that the loss or mutation of FBXW7 as seen in many types of cancer could lead to an abnormal elevation of Aurora B and result in deregulated mitosis, which accelerates cancer cell growth.

p53 plays an important role in mitotic checkpoint, but what its role is remains enigmatic. Aurora A is a Ser/Thr kinase involved in correcting progression of mitosis. Here, we show that p53 is a negative regulator for Aurora A. We found that p53 deficiency leads to Aurora A elevation. Ectopic expression of p53 or DNA damage-induced expression of p53 can suppress the expression of Aurora A. Mechanistic studies show that p53 is a negative regulator for Aurora A expression through both transcriptional and posttranslational regulation. p53 knockdown in cancer cells reduces the level of p21, which, in turn, increases the activity of CDK2 followed by induction of Rb1 hyperphosphorylation and its dissociation with transcriptional factor E2F3. E2F3 can bind to Aurora A gene promoter, potentiating Aurora A gene expression and p53 deficiency, enhancing the binding of E2F3 on Aurora A promoter. Also, p53 deficiency leads to decelerating Aurora A’s turnover rate, due to the fact that p53 deficiency causes the downregulation of Fbw7α, a component of E3 ligase of Aurora A. Consistently, p53 knockdown-mediated Aurora A elevation is mitigated when Fbw7α is ectopically expressed. Thus, p53-mediated Aurora A degradation requires Fbw7α expression. Significantly, inverse correlation between p53 and Aurora A elevation is translated into the deregulation of centrosome amplification. p53 knockdown leads to high percentages of cells with abnormal amplification of centrosome. These data suggest that p53 is an important negative regulator of Aurora A, and that loss of p53 in many types of cancer could lead to abnormal elevation of Aurora A and dysregulated mitosis, which provides a growth advantage for cancer cells.

Hyperglycemia during hyper-CVAD chemotherapy is associated with poor outcomes of acute lymphoblastic leukemia (ALL) (Cancer2004; 100:1179–85). The optimal clinical strategy to manage hyperglycemia during hyper-CVAD is unclear. To examine whether anti-diabetic pharmacotherapy can influence chemosensitivity of ALL cells, we examined the impacts of different anti-diabetic agents on ALL cell lines and patient samples. Pharmacologically achievable concentrations of insulin, aspart and glargine significantly increased the number of ALL cells, and aspart and glargine did so at lower concentrations than human insulin. In contrast, metformin and rosiglitazone significantly decreased the cell number. Human insulin and analogs activated AKT/mTOR signaling and stimulated ALL cell proliferation (as measured by flow cytometric methods), but metformin and rosiglitazone blocked AKT/mTOR signaling and inhibited proliferation. Metformin 500 μM and rosiglitazone 10 μM were found to sensitize Reh cells to daunorubicin, while aspart, glargine and human insulin (all at 1.25 mIU/L) enhanced chemoresistance. Metformin and rosiglitazone enhanced daunorubicin-induced apoptosis, while insulin, aspart and glargine antagonized daunorubicin-induced apoptosis. In addition, metformin increased etoposide-induced and L-asparaginase-induced apoptosis; rosiglitazone increased etoposide-induced and vincristine-induced apoptosis. In conclusion, our results suggest that use of insulins to control hyperglycemia in ALL patients may contribute to anthracycline chemoresistance, while metformin and thiazolidinediones may improve chemosensitivity to anthracycline as well as other chemotherapy drugs through their different impacts on AKT/mTOR signaling in leukemic cells. Our data suggest that the choice of anti-diabetic pharmacotherapy during chemotherapy may influence clinical outcomes in ALL.

Radiotherapy is the primary treatment for nasopharyngeal carcinoma (NPC), but radioresistance severely reduces NPC radiocurability. Here, we have established a radio-resistant NPC cell line, CNE-2R, and investigate the role of miRNAs in radioresistance. The miRNAs microarray assay reveals that miRNAs are differentially expressed between CNE-2R and its parental cell line CNE-2. We find that miR-205 is elevated in CNE-2R. A target prediction algorithm suggests that miR‑205 regulates expression of PTE N, a tumor-suppressor. Introducing miR-205 into CNE-2 cells suppresses PTE N protein expression, followed by activation of AKT, increased number of foci formation and reduction of cell apoptosis postirradiation. On the other hand, knocking down miR-205 in CNE-2R cells compromises the inhibition of PTE N and increases cell apoptosis. Significantly, immunohistochemistry studies demonstrate that PTE N is downregulated at late stages of NPC, and that miR-205 is significantly elevated followed the radiotherapy. Our data conclude that miR-205 contributes to radioresistance of NPC by directly targeting PTE N. Both miR-205 and PTE N are potential predictive biomarkers for radiosensitivity of NPC and may serve as targets for achieve successful radiotherapy in NPC.

The constitutive photomorphogenesis 9 signalosome (COP9 or CSN) is an evolutionarily conserved multiprotein complex found in plants and animals. Because of the homology between the COP9 signalosome and the 19S lid complex of the proteosome, COP9 has been postulated to play a role in regulating the degradation of polyubiquitinated proteins. Many tumor suppressor and oncogene products are regulated by ubiquitination- and proteosome-mediated protein degradation. Therefore, it is conceivable that COP9 plays a significant role in cancer, regulating processes relevant to carcinogenesis and cancer progression (e.g., cell cycle control, signal transduction and apoptosis). In mammalian cells, it consists of eight subunits (CSN1 to CSN8). The relevance and importance of some subunits of COP9 to cancer are emerging. However, the mechanistic regulation of each subunit in cancer remains unclear. Among the CSN subunits, CSN5 and CSN6 are the only two that each contain an MPN (Mpr1p and Pad1p N-terminal) domain. The deneddylation activity of an MPN domain toward cullin-RING ubiquitin ligases (CRL) may coordinate CRL-mediated ubiquitination activity. More recent evidence shows that CSN5 and CSN6 are implicated in ubiquitin-mediated proteolysis of important mediators in carcinogenesis and cancer progression. Here, we discuss the mechanisms by which some CSN subunits are involved in cancer to provide a much needed perspective regarding COP9 in cancer research, hoping that these insights will lay the groundwork for cancer intervention.

Tumor hypoxia directly promotes genomic instability and facilitates cell survival, resulting in tumors with a more aggressive phenotype. The proto-oncogene pim-1regulates apoptosis and the cell cycle by phosphorylating target proteins. Overexpression of Pim-1 can cause genomic instability and contribute to lymphomagenesis. It is not clear whether Pim-1 is involved in hypoxia-mediated tumor survival in solid tumors. Here, we show that hypoxia can stabilize Pim-1 by preventing its ubiquitin-mediated proteasomal degradation and can cause Pim-1 translocation from the cytoplasm to the nucleus. Importantly, overexpression of Pim-1 increases NIH3T3 cell transformation exclusively under hypoxic conditions, suggesting that Pim-1 expression under hypoxia may be implicated in the transformation process of solid tumors. Also, blocking Pim-1 function by introduction of dominant negative Pim-1 resensitizes pancreatic cancer cells to apoptosis induced by glucose-deprivation under hypoxia. Introduction of short interfering RNAs for Pim-1 also resensitizes cancer cells to glucose deprivation under hypoxic conditions, while forced overexpression of Pim-1 causes solid tumor cells to become resistant to glucose deprivation. Moreover, dominant negative Pim-1 reduces tumorigenicity in pancreatic cancer cells and HeLa xenograft mouse models. Together, our studies indicate that Pim-1 plays a distinct role in solid tumor formation in vivo, implying that Pim-1 may be a novel target for cancer therapy.

Cancer cells undergo mitosis frequently, and many mitotic regulators are aberrantly expressed in these cells. Members of the Aurora family of serine/threonine kinases are expressed during mitosis and carry out vital functions in chromosome alignment, segregation and cytokinesis. Here we review the functions of Aurora-B kinases in mitosis and summarize the current literature on Aurora-B kinase inhibitors. In the process of developing these inhibitors as anticancer drugs, the Aurora kinase inhibitors have also helped to advance our understanding of the role of Aurora kinases in mitosis. The mechanism of action and structure-activity relationship of a selective Aurora-B inhibitor are also discussed. The future may see mechanism guided design of chemotherapy combinations that include these cell-cycle phase-specific drugs. The therapeutic potential of Aurora-B inhibitors is promising.

The mechanisms of action of farnesyltransferase inhibitors (FTIs) involve Rheb and the phosphatidylinositide 3-kinase/Akt/mammalian target of rapamycin (mTOR) pathway. mTOR in particular plays a key role in the regulation of autophagy. Collectively, the literature suggests that FTIs very likely induce autophagy, but thus far there have been no reports that FTIs affect this process relevant to cancer cell biology. We hypothesized that FTIs can induce autophagy. In this study, we found that the FTIs manumycin A, FTI-276, and lonafarnib induced autophagy in two human cancer cell lines. We also found that neither inhibition of apoptosis with a pan-caspase inhibitor nor inhibition of autophagy increased the number of clones of lonafarnib-treated U2OS osteosarcoma cells that formed in soft agar. Although whether autophagy is a cell death or cell survival mechanism after FTI treatment remains unresolved, our data show that cancer cells apparently can shift between apoptosis and autophagy once they are committed to die after FTI treatment.

Anti-HER2 antibody trastuzumab (Herceptin®) has been used effectively to treat patients with breast cancer that overexpresses HER2. We have demonstrated that p27Kip1 upregulation is one of the key events that cause G1 arrest upon trastuzumab treatment. Here, we have examined the effect of trastuzumab on CDK2, Rb, E2F1, NPAT, histone H4, and their associated signaling pathway. Results showed that trastuzumab was able to dramatically inhibit the kinase activity and expression of CDK2, whereas the kinase activity and expression of CDK4 were not affected by trastuzumab treatment. Unlike the p27Kip1 upregulation that occurs primarily through post-translational mechanisms, CDK2 was mainly downregulated at a transcriptional level as shown by Northern blotting and real-time RT-PCR analyses. Trastuzumab decreased the kinase activity of cyclin E (because of CDK2 inhibition) although it had little effect on cyclin E protein level. Overexpression of wild-type cyclin E or its lower molecular weight forms did not influence the response to trastuzumab. Levels and activities of CDK6, cyclin A, and cyclin D1 were all suppressed by trastuzumab. As a result, Rb phosphorylations that associates with CDK2, cyclin E, CDK6, cyclin A, or cyclin D1 were substantially inhibited by trastuzumab. Trastuzumab decreased the DNA-binding activity of E2F1. Trastuzumab decreased the level of NPAT protein. Trastuzumab decreased the level of histone H4 mRNA. Blockade of PI3K pathway with LY294002 produced similar effects to trastuzumab treatment. Taken together, these results demonstrated that anti-HER2antibody trastuzumab inhibits CDK2, Rb phosphorylation, E2F1 activity, NPAT, and histone H4 via PI3K signaling pathway, which are essential to both DNA and histone synthesis during the progression of G1 phase to S phase of the cell cycle.

The mammalian cell cycle can be divided into four phases: G1 (gap phase 1), S (DNA synthesis), G2 (gap phase 2), and M (mitosis). Progression through each phase of the cell cycle is delicately controled by the activity of different cyclin-dependent kinases (CDKs) and their regulatory subunits known as cyclins. CDK2, CDK4, CDK6 and their associated cyclins control the G1 to S phase transition. The association of CDK4 or CDK6 with D-type cyclins is critical for G1 phase progression, whereas the CDK2-cyclin E complex is important for initiation of the S phase. Cancer can originate from dysregulation of these regulators. A variety of intrinsic and extrinsic signals were recently identified to regulate these G1 or G1/S CDKs and cyclins. Here we discuss the regulators of these protein kinases at different mechanistic level with a hope that these insights can be applied to develop therapeutic strategies for cancer treatment.

Cell cycle dysregulation is a critical feature of tumour cells. Numerous cell cycle regulators act either as oncogenes or tumour suppressors and their aberrations result in proliferative advantage for cancer cells. Many molecular targets and their use in either abrogating the growth advantage of oncogenic mediators or enhancing the growth suppressive activity of tumour suppressors, have been filed for patents. The molecular targets associated with cell cycle inhibition are of particular interest because they are potential therapeutic agents of promise in the control of inappropriate cellular proliferation. This review focuses on the recent discovery of potential molecular targets involved in cell cycle inhibition and their evaluation as therapeutic agents for cancers. In this review, the strategies employed to control oncogenesis and their possible clinical applications are discussed.

Women with unilateral breast carcinoma are at increased risk for developing contralateral disease. The clinical significance of bilateral breast carcinoma has not been fully defined, and the subset of patients who may benefit from medical or surgical risk-reduction intervention has not yet been characterized. The purpose of this study was to evaluate risk factors and outcomes for bilateral breast carcinoma. A subject group of 70 bilateral breast carcinoma patients (62% metachronous) was matched by age and survival interval with a control group of 70 unilateral breast carcinoma patients. Median follow-up was 103 months. Eighty-two percent of the unilateral patients and 80% of the bilateral patients had Stage I or II disease at diagnosis. Median age at presentation was 53 years. In the bilateral group, the contralateral cancer was diagnosed at the same or earlier stage than the first cancer in 87% of cases. Bilateral patients were significantly more likely to have multicentric disease and to have a positive family history for breast carcinoma compared with the unilateral group. There were no significant differences regarding history of exogenous hormone exposure, lobular histology, hormone-receptor status, or HER-2/neu expression. Five-year disease-free survival was 94% for the unilateral breast carcinoma patients and 91% for the bilateral breast carcinoma patients (P = 0.16). Survival for patients with bilateral breast carcinoma is similar to that of patients with unilateral disease; however, prophylactic risk-reduction intervention for the contralateral breast should be considered in patients who have multicentric unilateral disease or a positive family history for breast carcinoma. Cancer 2001;91:1845–53. © 2001 American Cancer Society.

Overexpression and activation of HER-2/neu, a proto-oncogene, play a pivotal role in cancer formation. Strong expression of HER-2/neuin cancers has been associated with poor prognosis. Reduced expression of p27Kip1, a cyclin-dependent kinase inhibitor, correlates with poor clinical outcome in many types of carcinomas. Because many cancers with the overexpression of HER-2/neuoverlap with those affected by reduced p27 expression, we studied the link between HER-2/neuoncogenic signals and p27 regulation. We found that down-regulation of p27 correlates with HER-2/neuoverexpression. To address the molecular mechanism of this inverse correlation, we found that reduction of p27 is caused by enhanced ubiquitin-mediated degradation, and the HER-2/Grb2/MAPK pathway is involved in the decrease of p27 stability. Also, HER-2/neuactivity causes mislocation of p27 andJun activation domain-binding protein 1 (JAB1), an exporter of p27, into the cytoplasm, thereby facilitating p27 degradation. These results reveal that HER-2/neusignals reduce p27 stability and thus present potential points for therapeutic intervention in HER-2/neu-associated cancers.

14-3-3 sigma, implicated in cell cycle arrest by p53, was cloned by expression cloning through cyclin-dependent kinase 2 (CDK2) association. 14-3-3 sigma shares cyclin-CDK2 binding motifs with different cell cycle regulators, including p107, p130, p21CIP1, p27KIP1, and p57KIP2, and is associated with cyclin·CDK complexesin vitroand in vivo. Overexpression of 14-3-3 sigma obstructs cell cycle entry by inhibiting cyclin-CDK activity in many breast cancer cell lines. Overexpression of 14-3-3 sigma can also inhibit cell proliferation and prevent anchorage-independent growth of these cell lines. These findings define 14-3-3 sigma as a negative regulator of the cell cycle progression and suggest that it has an important function in preventing breast tumor cell growth.

Lysogenization of mycobacteriophage L5 involves integration of the phage genome into the Mycobacterium smegmatischromosome. Integration occurs by a site-specific recombination event between a phage attachment site, attP, and a bacterial attachment site, attB, which is catalyzed by the phage-encoded integrase protein. DNase I footprinting reveals that L5 integrase binds to two types of sites within attPwhich span an unexpectedly large region of 413 bp: seven arm-type sites (P1 to P7) each of which correspond to a consensus sequence 5′-TGCaaCtcYy, and core-type sites at the points of strand exchange. Mutational analyses indicate that not all of the arm-type sites are required for integration, and that the P3 site and the rightmost pair of sites (P6 and P7) are dispensable for integration. We show that a 252 bp segment of attPDNA is sufficient for efficient integrative recombination and that intcan be provided in transfor simple and efficient transformation of the mycobacteria.

Commentary to:Effect of DNAzymes Targeting Akt1 on Cell Proliferation and Apoptosis in Nasopharyngeal CarcinomaLifang Yang, Lanbo Xiao, Xiaoqian Ma, Min Tang, Xinxiang Weng, Xue Chen, Lunquan Sun，Ya Cao

Farnesyltransferase inhibitors (FTIs) were designed to block the action of Ras oncoproteins which depend on posttranslational modification by adding a farnesyl isoprenoid membrane anchor.  However, off-target actions are believed to account for most of their antitumor activity.  We recently reported the induction of autophagy in cancer cells in a dose-dependent manner by FTIs.  We observed similar results of autophagy in a panel of tumor cell lines for the three FTIs tested.  Therefore, the induction of autophagy is very likely a pharmacological class effect of inhibition of farnesyltransferase.  In this addendum, we discuss the possible mechanisms underlying the induction of autophagy by FTIs, including reactive oxygen species-, DNA damage- and Ras-mediated pathways as alternatives to Rheb-mediated regulation of mTOR and autophagy.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Metformin is derived from galegine, a natural ingredient, and recent studies have suggested that metformin could enhance the antitumor effects of hormone ablative therapy or chemotherapy and reduce prostate cancer-specific mortality. Zyflamend is a combination of herbal extracts that reduces inflammation and comprises turmeric, holy basil, green tea, oregano, ginger, rosemary, Chinese goldthread, hu zhang, barberry, and basil skullcap. We propose a maintenance regimen with metformin and/or Zyflamend that targets cancer stem cells and the tumor microenvironment to keep the cancer dormant and prevent it from activation from dormancy. Herein, we report the clinical course of four patients who experienced a clinical response after treatment with metformin and/or Zyflamend.